Immunological compositions containing attenuated Histophilus somni

ABSTRACT

This disclosure provides attenuated  Histophilus somni  strains, compositions comprising same, and methods of production and use thereof. The attenuated strains may express lower or no levels of various virulence-associated genes, relative to the corresponding pathogenic bacteria Advantageously, the attenuated  Histophilus somni  strains may be administered orally, intranasally, intra-tracheally, or subcutaneously.

INCORPORATION BY REFERENCE

This application is a continuation of, and claims benefit of, U.S.application Ser. No. 14/668,656, filed on Mar. 25, 2015, now U.S. Pat.No. 9,592,283, which claims the benefit of U.S. provisional ApplicationNo. 61/970,195, filed on 25 Mar. 2014, the entire contents of which arehereby incorporated by reference herein. All other references citedherein are also herein incorporated by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is MER_13_225P_ST25. The text file is 17,356 KB; itwas created on Feb. 20, 2014; and it is being submitted electronicallyvia EFS-Web, concurrent with the filing of the specification.

FIELD OF THE INVENTION

The present invention relates generally to genetically engineered,attenuated bacterial vaccines, particularly those providing broad, safe,and effective protection to bovines against infections/disease caused byHistophilus somni (formerly Haemophilus somnus). The invention furtherrelates to methods of producing the attenuated bacteria, and to theidentification of nucleic acid variations that are associated withdecreased pathogenicity of the attenuated bacteria.

The invention accordingly relates to immunogenic or vaccine compositionscomprising the bacteria of the invention; e.g., live attenuatedbacteria. The bacteria also could be inactivated in the compositions;but it may be advantageous that the bacteria are live attenuatedHistophilus somni. The invention therefore further relates to methodsfor preparing and/or formulating such compositions; e.g., culturing orgrowing or propagating the bacteria on or in suitable medium, harvestingthe bacteria, optionally inactivating the bacteria, and optionallyadmixing with a suitable veterinarily or pharmaceutically acceptablecarrier, excipient, diluent or vehicle and/or an adjuvant and/orstabilizer. Thus, the invention also relates to the use of the bacteriain formulating such compositions.

BACKGROUND OF THE INVENTION

Bovine Respiratory Disease Complex (BRDC) consists of multiple microbialpathogens and contributes to substantial economic loss to the cattleindustry. Treatment costs including both preventative vaccination andmedication following an outbreak are estimated to be near $4 billion peryear (Griffin, 1997). Adding to the economic impact is the associatedloss in performance observed in animals diagnosed with BRDC; withmeasurable losses to average daily gain, body weight at harvest, andbeef quality grade (Babcock, 2010). Reports on specific monetary lossassociated with decreased performance vary; likely due to varying casedefinitions of BRDC, but are estimated between $40 (Fulton, 2002) andalmost $300 (Duff and Gaylean 2011) per animal. The performance loss hasalso been shown to significantly increase with the number of times ananimal requires treatment for BRDC (Fulton, 2002).

Histophilus somni (formerly Haemophilus somnus) has been identified as akey contributor to BRDC (Duff and Gaylean 2011). This gram-negativepleomorphic coccobacillus belonging to the family Pasterellacea(Korczak, 2004) makes up part of the normal microbiota of the upperrespiratory and urogenital tracts in cattle, sheep, and other ruminants(Ward, 2006). It is closely related to other bovine pathogens includingPasteurella multocida and Mannheimia haemolytica (both of which are alsoassociated with BRDC) as well as the human pathogens Haemophilus ducreyiand Haemophilus influenzae (Challacombe, 2007).

Estimates place the isolation rate of H. somni from the upperrespiratory tract of healthy calves as high as 50% with no clinicalmanifestations of disease; however animals diagnosed with BRDC have aneven higher isolation rate for the bacteria (Griffin, 2010). Understressful conditions or states of immunosuppression, H. somni maycolonize the lower respiratory tract, endocardium, or central nervoussystem and has been identified as an etiological agent in diversediseases such as pneumonia, endocarditis, arthritis, abortion,septicemia, and thromboembolic meningoencephalitis (TEME) (Ward, 2006).

At the time of slaughter, less than 15% of animals receiving propertreatment for BRDC (preventative vaccinations and appropriateantibiotics during an outbreak) show signs of lung lesions and theselesions involve less than 5% of the total lung (Griffin, 2010).Conversely, 50% of animals not receiving proper care display lunglesions at the time of slaughter, and these lesions may involve 15% ormore of the total lung (Griffin, 2010). In one field study of over10,000 animals, 459 calves (4.6%) died from disease of one form oranother. Of the mortalities in the study, 279 (60.8%) were shown to berelated to respiratory ailments, and of those with respiratoryinfections, 226 (81.0%) were associated with H. somni related pneumonia,pleurisy, or abscesses (Ribble, 1988). While antibiotic treatment may besuccessful in response to an H. somni infection, an increasingprevalence of antibiotic resistant field isolates is of concern (Duffand Gaylean, 2011). Preventative care by vaccination would be preferredas it is proactive rather than reactive and is much more cost effective.

Many H. somni vaccines are currently available from various animalhealth companies; however these vaccines are predominantly composed ofkilled bacterins and were licensed over thirty years ago with an aim inpreventing TEME. The use of these bacterin vaccines has been effectiveagainst TEME, however has been shown to have neutral or even negativeeffects on respiratory disease in feedlot cattle. Negative side-effectsinclude IgE induced anaphylactic shock and interactions when calvesinfected with Bovine Respiratory Syncytial Virus (BRSV) are vaccinated(Griffin, 2010). The decrease in prevalence of TEME and the emergence ofH. somni related pneumonia in the US and myocarditis in Canada beginningin the late 1980's, have led to a need for further investigation ofefficacious antigens for vaccine production (O'Toole, 2009).

H. somni related pneumonia is an economically significant condition forthe beef and dairy industries. There is little evidence of fieldefficacy in currently available vaccines, so the need for research intonext generation vaccine products is warranted.

SUMMARY OF THE INVENTION

An object of this invention is to provide attenuated bacteria as well asmethods for treatment and prophylaxis of infection by H. somni.

In an embodiment, the vaccines comprise attenuated H. somni, which areless pathogenic as compared to a more virulent strain. Comprehensivecomparative sequence analysis revealed the absence of importantvirulence factors in the attenuated H. somni strains, relative to morevirulent strains. Thus, it is an object of the invention to provideattenuated H. somni, which are lacking in virulence factors possessed bymore virulent H. somni strains.

As defined herein, the term “gene” will be used in a broad sense, andshall encompass both coding and non-coding sequences (i.e. upstream anddownstream regulatory sequences, promoters, 5′/3′ UTR, introns, andexons). Where reference to only a gene's coding sequence is intended,the term “gene's coding sequence” or “CDS” will be used interchangeablythroughout this disclosure. When a specific nucleic acid is discussed,the skilled person will instantly be in possession of all derivableforms of that sequence (mRNA, vRNA, cRNA, DNA, protein, etc.).

The invention further provides methods for inducing an immunological (orimmunogenic) or protective response against H. somni, as well as methodsfor preventing or treating H. somni, or disease state(s) caused by H.somni, comprising administering the attenuated H. somni, or acomposition comprising the attenuated H. somni to animals in needthereof. Moreover, kits comprising at least the attenuated H. somnistrain(s) and instructions for use are also provided.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof, to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification, includingreference to the accompanying figures, wherein:

FIG. 1 shows the average percent lung lesions for each isolate and dosetested in the challenge model. Groups with different letters aresignificantly different according to Student's t performed on thearcsine transformed data;

FIG. 2 shows the percent lung lesions observed for each calf in avaccination-challenge study where the vaccine was administeredintranasally. Bars of the same color represent the results for animalsvaccinated with the same isolate. The vaccination isolate is listedbelow the bars, and all animals were challenged with the same challengeisolate. Groups followed by the same letter(s) are not significantlydifferent according to Student's t performed on the arcsine transformeddata.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides nucleotide sequences and genes involvedin the attenuation of a microorganism, such as bacteria, for instance,H. somni, products (e.g., proteins, antigens, immunogens, epitopes)encoded by the nucleotide sequences, methods for producing suchnucleotide sequences, products, micro-organisms, and uses thereof, suchas for preparing vaccine or immunogenic compositions or for eliciting animmunological or immune response or as a vector, e.g., as an expressionvector (for instance, an in vitro or in vivo expression vector).

Mutations identified in nucleotide sequences and genes ofmicro-organisms produce novel and nonobvious attenuated mutants. Thesemutants are useful for the production of live attenuated immunogeniccompositions or live attenuated vaccines having a high degree ofimmunogenicity.

Identification of the mutations provides novel and nonobvious nucleotidesequences and genes, as well as novel and nonobvious gene productsencoded by the nucleotide sequences and genes.

In an embodiment, the invention provides an attenuated H. somni straincapable of providing a safe and effective immune response in cattleagainst H. somni or diseases caused by H. somni.

In an aspect, the invention provides bacteria containing an attenuatingmutation in a nucleotide sequence or a gene wherein the mutationmodifies the biological activity of a polypeptide or protein encoded bya gene, resulting in attenuated virulence of the bacteria.

In particular, the present invention encompasses attenuated H. somnistrains and vaccines comprising the same, which elicit an immunogenicresponse in an animal, particularly the attenuated H. somni strains thatelicit, induce or stimulate a response in a bovine.

Particular H. somni attenuated strains of interest have mutations ingenes, relative to virulent strains. It is recognized that, in additionto strains having the disclosed mutations, attenuated strains having anynumber of mutations in the disclosed virulence genes can be used in thepractice of this invention.

In another aspect, the novel attenuated H. somni strains are formulatedinto safe, effective vaccine against H. somni and infections/diseasescause by H. somni.

In an embodiment, the attenuated H. somni strain is capable of providinga safe and effective immune response in a bovine against H. somni ordiseases caused by H. somni.

In a particular embodiment, the attenuated strain is lacking one orseveral virulence genes, relative to an otherwise genetically similarvirulent strain. In an embodiment, the attenuated strain lacks and/ordoes not express the glycoside hydrolase family protein (HSM_1160) andthe lipoprotein (HSM_1714). Absent genes that may also be contribute tothe strain's attenuated phenotype include: multicopper oxidase type 3(HSM_1726) and a TetR family transcriptional regulator (HSM_1734). In aneven more particular embodiment, the attenuated strain is the same asthat deposited in the ATCC under the designation PTA-121029.

In an embodiment, the strain may be administered intranasally orsubcutaneously.

In another aspect, the invention encompasses an immunologicalcomposition comprising the disclosed attenuated H. somni strains. Thecomposition may further comprise a pharmaceutically or veterinaryacceptable vehicle, diluent or excipient.

In an embodiment, the composition may provide a safe and protectiveimmune response in bovine against subsequent virulent H. somnichallenge.

In still another embodiment, the composition may comprise a geneticallyengineered, non-naturally-occurring, attenuated H. somni strain,suitable for use in a safe and effective vaccine formulation, and havingat least the following genes mutated, including completely deleted, toeliminate the ability of the genes to express their cognate geneproduct: HSM_0270, HSM_0338, HSM_0377, HSM_0598, HSM_0708, HSM_0749,HSM_0953, HSM_1160, HSM_1191, HSM_1257, HSM_1426, HSM_1616, HSM_1624,HSM_1728, HSM_1730, HSM_1734, HSM_1736, HSM_1737, HSM_1741, HSM_1793 andHSM_1889. In a particular embodiment, the engineered H. somni strain hasan identical attenuated phenotype as compared with TK #4.

In a related embodiment, the composition may comprise a geneticallyengineered, non-naturally-occurring, attenuated H. somni strain,suitable for use in a safe and effective vaccine formulation, and havinga sufficient number of the following genes mutated, including completelydeleted, to eliminate the ability of the genes to express their cognategene product: HSM_0270, HSM_0338, HSM_0377, HSM_0598, HSM_0708,HSM_0749, HSM_0953, HSM_1160, HSM_1191, HSM_1257, HSM_1426, HSM_1616,HSM_1624, HSM_1728, HSM_1730, HSM_1734, HSM_1736, HSM_1737, HSM_1741,HSM_1793 and HSM_1889. In a particular embodiment, the engineered H.somni strain has an identical attenuated phenotype as compared with TK#4.

In another embodiment, the composition may comprise a geneticallyengineered, non-naturally-occurring, attenuated H. somni strain,suitable for use in a safe and effective vaccine formulation, and havingat least the following genes mutated, including completely deleted, toeliminate the ability of the gene to express its cognate gene product:HSM_0077, HSM_0270, HSM_0708, HSM_0975, HSM_1191, HSM_1257, HSM_1448,HSM_1542, HSM_1571, HSM_1624, HSM_1714, HSM_1726, HSM_1728, HSM_1730,HSM_1734, HSM_1736, HSM_1737, HSM_1741 and HSM_1793. In a particularembodiment, the engineered H. somni strain has an identical attenuatedphenotype as compared with TK #42.

In a related embodiment, the composition may comprise a geneticallyengineered, non-naturally-occurring, attenuated H. somni strain,suitable for use in a safe and effective vaccine formulation, and havinga sufficient number of the following genes mutated, including completelydeleted, to eliminate the ability of the gene to express its cognategene product: HSM_0077, HSM_0270, HSM_0708, HSM_0975, HSM_1191,HSM_1257, HSM_1448, HSM_1542, HSM_1571, HSM_1624, HSM_1714, HSM_1726,HSM_1728, HSM_1730, HSM_1734, HSM_1736, HSM_1737, HSM_1741 and HSM_1793.In a particular embodiment, the engineered H. somni strain has anidentical attenuated phenotype as compared with TK #42.

In another embodiment, the composition may comprise a geneticallyengineered, non-naturally-occurring, attenuated H. somni strain,suitable for use in a safe and effective vaccine formulation, and havingat least the following genes mutated, including completely deleted, toeliminate the ability of the gene to express its cognate gene product:HSM_0268, HSM_0270, HSM_0274, HSM_0598, HSM_0708, HSM_0749, HSM_0938,HSM_1022, HSM_1160, HSM_1191, HSM_1212, HSM_1257, HSM_1542, HSM_1571,HSM_1667, HSM_1728, HSM_1730, HSM_1736, HSM_1737, HSM_1741, HSM_1793 andHSM_1889. In a particular embodiment, the engineered H. somni strain hasan identical attenuated phenotype as compared with TK #34.

In a related embodiment, the composition may comprise a geneticallyengineered, non-naturally-occurring, attenuated H. somni strain,suitable for use in a safe and effective vaccine formulation, and havinga sufficient number of the following genes mutated, including completelydeleted, to eliminate the ability of the gene to express its cognategene product: HSM_0268, HSM_0270, HSM_0274, HSM_0598, HSM_0708,HSM_0749, HSM_0938, HSM_1022, HSM_1160, HSM_1191, HSM_1212, HSM_1257,HSM_1542, HSM_1571, HSM_1667, HSM_1728, HSM_1730, HSM_1736, HSM_1737,HSM_1741, HSM_1793 and HSM_1889. In a particular embodiment, theengineered H. somni strain has an identical attenuated phenotype ascompared with TK #34.

Now that Applicants have disclosed these sufficient sets of attenuatinggene deletions, the skilled person will appreciate that only non-routineworks remains to determine which sub-combinations of these genedeletions are necessary to produce comparably- orequivalently-attenuated H. somni vaccine strains.

In another embodiment, the composition may further comprise at least oneadditional antigen associated with or derived from a bovine pathogenother than H. somni.

In an embodiment, the at least one or more additional antigen(s) iscapable of eliciting in a cattle an immune response against H. somni,bovine respiratory disease complex (BRDC), bovine respiratory syncytialvirus (BRSV), bovine viral diarrhea (BVD), bovine parainfluenza 3 (PI3),infectious bovine rhinotracheitis (IBR), bovine herpesvirus-1 (BHV-1),bluetongue disease virus (BTV), or any other pathogen capable ofinfecting and causing illness or susceptibility to illness in a bovine.

In another aspect, the invention provides a method of vaccinating ananimal comprising administration of at least one of the disclosedimmunological compositions comprising the attenuated H. somni strains.

In an embodiment, the H. somni vaccines further comprise an adjuvant. Ina particular embodiment, the adjuvant comprises whole bacteria and/orbacteria, including clostridium, H. somni, Mannheimia, Pasteurella,Histophilus, Salmonella, Escherichia coli, or combinations and/orvariations thereof. In several embodiments, the adjuvant increases theanimal's production of IgM, IgG, IgA, and/or combinations thereof.

In another embodiment, the invention provides an attenuated Histophilussomni (H. somni) strain, which is capable of providing a safe andeffective immune response in a bovine animal against H. somni, ordiseases caused by H. somni; wherein the attenuated strain lacks, in itsgenomic sequence, a minimum number of virulence factor-encoding genes,relative to a reference virulent H. somni strain, to render theattenuated strain incapable of causing infection in the bovine animal.

In an embodiment, the reference virulent strain comprises a genomic DNAsequence, which encodes at least about 99% of the same genes as does thesequence as set forth in SEQ ID NO:2. In other embodiments, theattenuated strain encodes at least about 99% of the same genes as doesthe sequence as set forth in SEQ ID NO:1, 3, 4, or 5. In a particularembodiment, the attenuated strain(s) expresses any virulence factor geneat a level about equal to, or lower than (including an undetectablelevel), the level of the corresponding virulence gene expressed by anattenuated strain having for its genome the sequence as set forth in SEQID NO:1, 3, 4, or 5.

In a particular embodiment, the attenuated strain lacks at least about99% of the same genes, relative to H. somni strain 2336, as does H.somni strain #4. In this embodiment, strain 2336 comprises a genomicsequence, which encodes at least about 99% of the same genes as does thesequence as set forth in SEQ ID NO:6; and strain #4 comprises a genomicsequence, which encodes at least about 99% of the same genes as does thesequence as set forth in SEQ ID NO:1.

In a particular embodiment, the attenuated strain is the #4 isolate(i.e. TK #4), which is deposited at the ATCC under the designationPTA-121029. In another embodiment, the attenuated strain is the #42isolate (i.e. TK #42), which is deposited at the ATCC under thedesignation PTA-121030.

In another aspect, the invention provides immunological compositionscomprising any of the attenuated strains described herein. In anembodiment, the composition comprises an attenuated strain, whichencodes at least about 99% of the same genes as does the sequence as setforth in SEQ ID NO:1, 3, 4 or 5.

In one particular embodiment, the attenuated strain encodes at leastabout 99% of the same genes as does the sequence as set forth in SEQ IDNO:1. In a related embodiment, the attenuated strain encodes all thesame genes as does the sequence as set forth in SEQ ID NO:1.

In one embodiment, the attenuated strain encodes at least about 99% ofthe same genes as does the sequence as set forth in SEQ ID NO:4. In arelated embodiment, the attenuated strain encodes all the same genes asdoes the sequence as set forth in SEQ ID NO:4.

In still another embodiment, the attenuated strain encodes at leastabout 99% of the same genes as does the sequence as set forth in SEQ IDNO:3. In a related embodiment, the attenuated strain encodes all thesame genes as does the sequence as set forth in SEQ ID NO:3.

In yet another embodiment, the attenuated strain encodes at least about99% of the same genes as does the sequence as set forth in SEQ ID NO:5.In related embodiment, the attenuated strain encodes all the same genesas does the sequence as set forth in SEQ ID NO:5.

In an embodiment, the immunological composition further comprises apharmaceutically or veterinary acceptable vehicle, diluent or excipient.

In a particular embodiment, immunological composition is capable ofeliciting in a bovine animal a protective immune response, whichprotects the bovine against a subsequent exposure to a virulent H. somnistrain.

In another embodiment, the immunological composition further comprisesat least one or more additional antigen, which is capable of elicitingin a bovine animal a pathogen-specific immune response. In a particularembodiment, the at least one or more additional antigen elicits in thebovine an immune response sufficient to protect the animal from asubsequent exposure to the pathogen from which the antigen was derived.Thus, if a bovine PI3 antigen is included as a component of theimmunological composition, the composition should elicit a protectiveimmune response.

In another embodiment, the additional antigen is capable of eliciting ina bovine animal an immune response, which will enhance the animal'simmune response against a subsequent exposure to Bovine RespiratoryDisease Complex, BRSV, BVD, PI3 or any other pathogen capable ofinfecting and causing illness or susceptibility to illness in a bovineanimal. For example, a BRSV antigen will provide protection against asubsequent exposure to virulent BRSV.

In another aspect, the invention provides a method of vaccinating abovine animal comprising the step of administering to the bovine animalat least one dose of an immunological composition as herein described.

By “antigen” or “immunogen” means a substance that induces a specificimmune response in a host animal. The antigen may comprise a wholeorganism, killed, attenuated or live; a subunit or portion of anorganism; a recombinant vector containing an insert with immunogenicproperties; a piece or fragment of DNA capable of inducing an immuneresponse upon presentation to a host animal; a polypeptide, an epitope,a hapten, or any combination thereof. Alternately, the immunogen orantigen may comprise a toxin or antitoxin.

The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer can be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

The term “immunogenic or antigenic polypeptide” as used herein includespolypeptides that are immunologically active in the sense that onceadministered to the host, it is able to evoke an immune response of thehumoral and/or cellular type directed against the protein. Preferablythe protein fragment is such that it has substantially the sameimmunological activity as the total protein. Thus, a protein fragmentaccording to the invention comprises or consists essentially of orconsists of at least one epitope or antigenic determinant. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the protein, analogs thereof, or immunogenicfragments thereof. By “immunogenic fragment” is meant a fragment of aprotein which includes one or more epitopes and thus elicits theimmunological response described above. Such fragments can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes maybe determined by e.g., concurrently synthesizing large numbers ofpeptides on solid supports, the peptides corresponding to portions ofthe protein molecule, and reacting the peptides with antibodies whilethe peptides are still attached to the supports. Such techniques areknown in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysenet al., 1984; Geysen et al., 1986. Similarly, conformational epitopesare readily identified by determining spatial conformation of aminoacids such as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Methodsespecially applicable to the proteins of T. parva are fully described inPCT/US2004/022605 incorporated herein by reference in its entirety.

As discussed herein, the invention encompasses active fragments andvariants of the antigenic polypeptide. Thus, the term “immunogenic orantigenic polypeptide” further contemplates deletions, additions andsubstitutions to the sequence, so long as the polypeptide functions toproduce an immunological response as defined herein. The term“conservative variation” denotes the replacement of an amino acidresidue by another biologically similar residue, or the replacement of anucleotide in a nucleic acid sequence such that the encoded amino acidresidue does not change or is another biologically similar residue. Inthis regard, particularly preferred substitutions will generally beconservative in nature, i.e., those substitutions that take place withina family of amino acids. For example, amino acids are generally dividedinto four families: (1) acidic-aspartate and glutamate; (2)basic-lysine, arginine, histidine; (3) non-polar-alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar-glycine, asparagine, glutamine, cystine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another hydrophobicresidue, or the substitution of one polar residue for another polarresidue, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like; or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid that will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe reference molecule but possessing minor amino acid substitutionsthat do not substantially affect the immunogenicity of the protein are,therefore, within the definition of the reference polypeptide. All ofthe polypeptides produced by these modifications are included herein.The term “conservative variation” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite”. Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest. Preferably,the host will display either a therapeutic or protective immunologicalresponse such that resistance to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms and/or clinicaldisease signs normally displayed by an infected host, a quicker recoverytime and/or a lowered viral titer in the infected host.

By “animal” is intended mammals, birds, and the like. Animal or host asused herein includes mammals and human. The animal may be selected fromthe group consisting of equine (e.g., horse), canine (e.g., dogs,wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domesticcats, wild cats, other big cats, and other felines including cheetahsand lynx), ovine (e.g., sheep), bovine (e.g., cattle, calves, steers,bulls), porcine (e.g., pig), avian (e.g., chicken, duck, goose, turkey,quail, pheasant, parrot, finches, hawk, crow, ostrich, emu andcassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape),ferrets, seals, and fish. The term “animal” also includes an individualanimal in all stages of development, including newborn, embryonic andfetal stages.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a”, “an”, and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicate otherwise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

Compositions

The present invention relates to a H. somni vaccine or composition whichmay comprise an attenuated H. somni strain and a pharmaceutically orveterinarily acceptable carrier, excipient, or vehicle, which elicits,induces or stimulates a response in an animal.

The term “nucleic acid” and “polynucleotide” refers to RNA or DNA thatis linear or branched, single or double stranded, or a hybrid thereof.The term also encompasses RNA/DNA hybrids. The following arenon-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs, uracyl, other sugars andlinking groups such as fluororibose and thiolate, and nucleotidebranches. The sequence of nucleotides may be further modified afterpolymerization, such as by conjugation, with a labeling component. Othertypes of modifications included in this definition are caps,substitution of one or more of the naturally occurring nucleotides withan analog, and introduction of means for attaching the polynucleotide toproteins, metal ions, labeling components, other polynucleotides orsolid support. The polynucleotides can be obtained by chemical synthesisor derived from a microorganism.

The term “gene” is used broadly to refer to any segment ofpolynucleotide associated with a biological function. Thus, genesinclude introns and exons as in genomic sequence, or just the codingsequences as in cDNAs and/or the regulatory sequences required for theirexpression. For example, gene also refers to a nucleic acid fragmentthat expresses mRNA or functional RNA, or encodes a specific protein,and which includes regulatory sequences.

An “isolated” biological component (such as a nucleic acid or protein ororganelle) refers to a component that has been substantially separatedor purified away from other biological components in the cell of theorganism in which the component naturally occurs, for instance, otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinanttechnology as well as chemical synthesis.

The term “conservative variation” denotes the replacement of an aminoacid residue by another biologically similar residue, or the replacementof a nucleotide in a nucleic acid sequence such that the encoded aminoacid residue does not change or is another biologically similar residue.In this regard, particularly preferred substitutions will generally beconservative in nature, as described above.

The term “recombinant” means a polynucleotide with semisynthetic, orsynthetic origin which either does not occur in nature or is linked toanother polynucleotide in an arrangement not found in nature.

“Heterologous” means derived from a genetically distinct entity from therest of the entity to which it is being compared. For example, apolynucleotide may be placed by genetic engineering techniques into aplasmid or vector derived from a different source, and is a heterologouspolynucleotide. A promoter removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous promoter.

The polynucleotides of the invention may comprise additional sequences,such as additional encoding sequences within the same transcriptionunit, controlling elements such as promoters, ribosome binding sites,5′UTR, 3′UTR, transcription terminators, polyadenylation sites,additional transcription units under control of the same or a differentpromoter, sequences that permit cloning, expression, homologousrecombination, and transformation of a host cell, and any such constructas may be desirable to provide embodiments of this invention.

Methods of Use and Article of Manufacture

The present invention includes the following method embodiments. In anembodiment, a method of vaccinating an animal comprising administering acomposition comprising an attenuated H. somni strain and apharmaceutical or veterinarily acceptable carrier, excipient, or vehicleto an animal is disclosed. In one aspect of this embodiment, the animalis a bovine.

In one embodiment of the invention, a prime-boost regimen can beemployed, which is comprised of at least one primary administration andat least one booster administration using at least one commonpolypeptide, antigen, epitope or immunogen. Typically the immunologicalcomposition or vaccine used in primary administration is different innature from those used as a booster. However, it is noted that the samecomposition can be used as the primary administration and the boosteradministration. This administration protocol is called “prime-boost”.

A prime-boost regimen comprises at least one prime-administration and atleast one boost administration using at least one common polypeptideand/or variants or fragments thereof. The vaccine used inprime-administration may be different in nature from those used as alater booster vaccine. The prime-administration may comprise one or moreadministrations. Similarly, the boost administration may comprise one ormore administrations.

The dose volume of compositions for target species that are mammals,e.g., the dose volume of cattle or bovine compositions, based onbacterial antigens, is generally between about 0.1 to about 2.0 ml,between about 0.1 to about 1.0 ml, and between about 0.5 ml to about 1.0ml.

The efficacy of the vaccines may be tested about 3 to 5 weeks after thelast immunization by challenging animals, such as bovine, with avirulent, heterologous strain of H. somni. The animal may be challengedintra-nasally, intra-tracheally, and/or trans-tracheally. Samples fromnasal passages, trachea, lungs, brain, and/or mouth may be collectedbefore and post-challenge and may be analyzed for the presence of H.somni-specific antibody.

The compositions comprising the attenuated viral strains of theinvention used in the prime-boost protocols are contained in apharmaceutically or veterinary acceptable vehicle, diluent or excipient.The protocols of the invention protect the animal from H. somni and/orprevent disease progression in an infected animal.

The various administration is preferably a one-shot dosage, but multipledosages could be carried out 1 to 6 weeks apart. A preferred timeinterval is 2 to 3 weeks, and an annual booster is also envisioned. Inan embodiment, the compositions are administered to calves that arebetween about 5 to about 6 weeks old. In another embodiment, the calvesmay be about 3 to about 4 weeks old.

It should be understood by one of skill in the art that the disclosureherein is provided by way of example and the present invention is notlimited thereto. From the disclosure herein and the knowledge in theart, the skilled artisan can determine the number of administrations,the administration route, and the doses to be used for each injectionprotocol, without any undue experimentation.

Another embodiment of the invention is a kit for performing a method ofeliciting or inducing an immunological or protective response against H.somni in an animal comprising an attenuated H. somni immunologicalcomposition or vaccine and instructions for performing the method ofdelivery in an effective amount for eliciting an immune response in theanimal.

Another embodiment of the invention is a kit for performing a method ofinducing an immunological or protective response against H. somni in ananimal comprising a composition or vaccine comprising an attenuated H.somni strain of the invention, and instructions for performing themethod of delivery in an effective amount for eliciting an immuneresponse in the animal.

The pharmaceutically or veterinarily acceptable carriers or vehicles orexcipients are well known to one skilled in the art. For example, apharmaceutically or veterinarily acceptable carrier or vehicle orexcipient can be a 0.9% NaCl (e.g., saline) solution or a phosphatebuffer. Other pharmaceutically or veterinarily acceptable carrier orvehicle or excipients that can be used for methods of this inventioninclude, but are not limited to, poly-(L-glutamate) orpolyvinylpyrrolidone. The pharmaceutically or veterinarily acceptablecarrier or vehicle or excipients may be any compound or combination ofcompounds facilitating the administration of the attenuated bacteria.Doses and dose volumes are herein discussed in the general descriptionand can also be determined by the skilled artisan from this disclosureread in conjunction with the knowledge in the art, without any undueexperimentation.

Though the disclosed results were obtained without the use of anadjuvant, the immunological compositions and vaccines may furthercomprise or consist essentially of an appropriate adjuvant. Suitableadjuvants for use in the practice of the present invention are (1)polymers of acrylic or methacrylic acid, maleic anhydride and alkenylderivative polymers, (2) immunostimulating sequences (ISS), such asoligodeoxyribonucleotide sequences having one or more non-methylated CpGunits (Klinman et al., 1996; WO98/16247), (3) an oil in water emulsion,such as the SPT emulsion described on page 147 of “Vaccine Design, TheSubunit and Adjuvant Approach” published by M. Powell, M. Newman, PlenumPress 1995, and the emulsion MF59 described on page 183 of the samework, (4) cationic lipids containing a quaternary ammonium salt, e.g.,DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7)saponin or (8) other adjuvants discussed in any document cited andincorporated by reference into the instant application, or (9) anycombinations or mixtures thereof.

In an embodiment, adjuvants include those which promote improvedabsorption through mucosal linings. Some examples include MPL, LTK63,toxins, PLG microparticles and several others (Vajdy, M. Immunology andCell Biology (2004) 82, 617-627). In an embodiment, the adjuvant may bea chitosan (Van der Lubben et al. 2001; Patel et al. 2005; Majithiya etal. 2008; U.S. Pat. No. 5,980,912).

REFERENCES

-   Alekshun, M. N. and Levy, S. B. 2007. Molecular mechanisms of    antibacterial multidrug resistance. Cell. 128(6):1037-1050.-   Asgarali, A. Stubbs, K. A., Oliver, A., Vocaldo, D. J., and    Mark, B. L. 2009. Inactivation of the Glycoside Hydrolase NagZ    Attenuates Antipseudomonal β-Lactam Resistance in Pseudomonas    aeruginosa. Antimicrobial Agents and Chemotherapy. 53(6):2274-2282.-   Aubry, C et al. 2011. OatA, a peptidoglycan 0-acetyltransferase    involved in Listeria monocytogenes immune escape, is critical for    virulence. J. Infect. Dis. 204(5):731-740.-   Babcock, A. H. 2010. Epidemiology of Bovine Respiratory Disease and    Mortality in Commercial Feedlots. Kansas State University (doctoral    dissertation).-   Berghaus, L. J., Corbeil, L. B., Berghaus, R. D., Kalina, W. V.,    Kimball, R. A., and Gershwin, L. J. 2006. Effects of dual    vaccination for bovine respiratory syncytial virus and Haemophilus    somnus on immune responses. Vaccine. 24:6018-6027.-   Brown, N. L., Stoyanov, J. V., Kidd, S. P., and Hobman J. L. 2003.    The MerR family of transcriptional regulators. FEMS Microbiology    Reviews. 27:145-163.-   Challacombe et al. 2007. Complete Genome Sequence of Haemophilus    somnus (Histophilus somni) strain 129Pt and Comparison to    Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd. Journal    of Bacteriology 189(5); p 1890-1898.-   Corbeil, L. B. et. al. 1985. Serum susceptibility of Haemophilus    somnus from bovine clinical cases and carriers. Journal of Clinical    Microbiology. 22(2):192-198.-   Cox, A. D., Howard, M. D., Brisson, J.-R., Van Der Zwan M.,    Thibault, P., Perry, M. B., and Inzana, T. J. 1998. Structural    analysis of the phase-variable lipooligosaccharide from Haemophilus    somnus strain 738. European Journal of Biochemistry.    97:253(2):507-516.-   Daines D. A., Jarisch J., and Smith A. L. 2004. Identification and    characterization of a nontypeable Haemophilus influenzae putative    toxin-antitoxin locus. BMC Microbiol. July 26; 4:30.-   Duff and Gaylean 2011. Recent Advances in Management of Highly    Stressed, Newly Received Feedlot Cattle. Journal of Animal Science.    85; p 823-840.-   Garbe, J. and Collin, M. 2012. Bacterial Hydrolysis of Host    Glycoproteins—Powerful Protein Modification and Efficient Nutrient    Acquisition. Journal of Innate Immunity. 4:121-131.-   Fulton, R. W. et al. 2002. Evaluation of Health Status of Calves and    the Impact on Feedlot Performances: Assessment of a Retained    Ownership Program for Postweaning Calves. Can. J. Vet. Res. 66,    173-180.-   Geertsema, R. S., Worby, C., Kruger, R. P., Tagawa, Y., Russo, R.,    Herdman, D. S., Lo, K. Kimball, R. A., Dixon, J., and    Corbeil, L. B. 2008. Protection of mice against H. somni septicemia    by vaccination with recombinant immunoglobulin binding protein    subunits. Vaccine. 26:4506-4512.-   Gershwin, L. J., Berghaus, L. J. Arnold, K., Anderson, M. L., and    Corbeil, L. B. 2005. Immune mechanisms of pathogenic synergy in    concurrent bovine pulmonary infection with Haemophilus somnus and    bovine respiratory syncytial virus. 2005. Veterinary Immunology and    Immunopathology. 107:119-130.-   Griffin, D. 2010, Bovine Pasteurellosis and other Bacterial    Infections of the Respiratory Tract. Vet. Clin. North Am. Food Anim.    Pract. 26(1); p 57-71.-   Griffin, D. 1997. Economic Impact Associated with Respiratory    Disease in Beef Cattle. Vet. Clin. North Am. Food Anim. Pract. 13; p    367-377.-   Guzmán-Brambila, C et al. 2012. Two Outer Membrane Lipoproteins    from H. somni are Immunogenic in Rabbits and Sheep and Induce    Protection against Bacterial Challenge in Mice. Clinical and Vaccine    Immunology. 19(11):1826-1832.-   Huston, W. M., Jennings, M. P., McEwan, A. G. 2002. The multicopper    oxidase of Pseudomonas aeruginosa is a ferroxidase with a central    role in iron acquisition. Molecular Microbiology. 45(6):1741-1750.-   Kahler, C. et. al. 1996. IgtF and RfaK constitute the    lipooligosaccharide ice (inner core extension) biosynthesis operon    of N. meningitidis. J. Bacteriology. December 6677-6684.-   Kjos, M., Snipen, L., Salehian, Z., Nes, I. F., and    Diep, D. B. 2010. The Abi proteins and their involvement in    bacteriocin self-immunity. Journal of Bacteriology.    192(8):2068-2076.-   Korczak et. al. 2004. Phylogeny of the Family Pasteurellaceae based    on rpoB sequences. International Journal of Systemic and    Evolutionary Microbiology. 54; p 1393-1399.-   Liu, T. et. al. 2008. Immunological responses against S. enterica    serovar Typhimurium Braun lipoprotein and lipid A mutant strains in    Swiss-Webster mice: Potential use as live-attenuated vaccines.    Microbial Pathogenesis. 44(3):224-237.-   Perera, I. C. and Grove, A. 2010. Molecular Mechanisms of    Ligand-Mediated Attenuation of DNA Binding by MarR Family    Transcriptional Regulators. JMCB. 2(5):243-254.-   O'Toole et al. 2009. Diagnostic Exercise: Myocarditis due to    Histophilus somni in Feedlot and Backgrounded Cattle. Veterinary    Pathology. 46; p 1015-1017.-   Ramos, J. L., Martinez-Bueno, M., Molina-Henares, A. J., Terán, W.,    Watanabe, K., Zhang, X., Trinidad Gallegos, M., Brennan, R., and    Tobes, R. 2005. The TetR family of Transcriptional Repressors. MMBR.    69:326-356.-   Ribble et al. 1988. Efficacy of Immunization of Feedlot Cattle with    a Commercial Haemophilus somnus bacterin. Canadian Journal of    Veterinary Research. 52; p 191-198.-   Schafer, A et al. 1994. Cloning and characterization of a DNA region    encoding a stress-sensitive restriction system from Corynebacterium    glutamicum ATCC 13032 and analysis of its role in intergenic    conjugation with E. coli. J. Bacteriology. 176(23):7309-7319.-   Ward et al. 2006. Haemophilus somnus (Histophilus somni) in Bighorn    Sheep. Canadian Journal of Veterinary Research. 70; p. 34-42.

The invention will now be further described by the followingnon-limiting examples.

EXAMPLES

The literature has reported on a number of H. somni virulence factors.No single virulence factor seems to dominate the role of the pathogen,but instead, several factors appear to act in concert to make a givenisolate virulent. Seven virulence factors were identified: DR2 (a directrepeat that contains a conserved cytotoxic Fic motif within the IbpAdomain involved in resistance to serum), hsst-I(CMP-Neu5Ac-β-Gal-α-(2-3)-sialyltransferase, where sialylation of thelipooligosaccharide (LOS) has been shown to inhibit antibody binding),lob2b (encoding a glycotransferase involved in LOS synthesis), nan lyase(N-acetylneuraminate lyase, involved in sialic acid metabolism), nanepimerase (N-acetylmannosamine-6-phosphate-2-epimerase, involved in thetransport and metabolism of carbohydrates), luxS (involved in AI-2quorum sensing), and uspE (a universal stress protein necessary for cellmotility and aggregation in biofilm formation). Primers were designed toamplify each of these genes and PCR reaction parameters were optimized.

Fifty isolates were selected for screening. The criteria for isolateselection was they had to be isolated within approximately one year ofthe start of this study and had to be from diverse geographicallocations or from cases of particular interest with high possiblegenotypic diversity. The purpose of this was to look at the most currentisolates indicative of H. somni in the field and also to try and screenas much diversity as possible. Isolates were evaluated on their growthand colony appearance, in addition to carrying out the PCR reactionsdefined for each virulence factor of interest. The PCR reactions werevisualized on a 1% agarose gel. Following the selection of isolatesbased on growth, colony appearance, and PCR results, animal models wereidentified and used to evaluate isolates of interest.

Example 1—H. somni Mouse Challenge Model Development

Based on growth, geographical diversity, and sequence information, 21isolates were selected for evaluation in mice (Table 1).

TABLE 1 Isolates evaluated for a naturally occurring H. somni vaccinecandidate. Isolate # Case # Location SEQ ID NO TK #1 12-0137-7Grandview, ID TK #2 12-0137-8 Grandview, ID TK #3 12-0137-9 Grandview,ID TK #4 12-0137-4 Grandview, ID 1 TK #5 12-0137-6 Grandview, ID TK #1412-0137-13 Grandview, ID TK #15 12-0137-11 Grandview, ID TK #2111-4134-1 Marshall, MN 2 TK #22 11-4601-1 Great Bend, KS TK #2411-2909-2 Carlisle, KY TK #28 11-0125-1 Plainview, TX 3 TK #30 11-3169-2Las Animas, CO TK #33 11-3454-4 Fairmont, MN TK #34 11-3452-2 Fairmont,MN 5 TK #37 12-0370-1 Cresco, IA TK #40 11-0012-1 Gettysburg, SD TK #4111-4378-2 Gettysburg, SD TK #42 11-0141-1 Parkston, SD 4 TK #4411-0263-1 Mesquite, NV TK #47 11-4312-1 Sioux Center, IA 2336 2336Public WT (Control) 6

Isolates stored at −80° C. were streak-plated onto Columbia+5% SheepBlood agar plates (CSBA) (Becton Dickinson, Franklin Lakes, N.J.). Theplates were incubated at 37° C. with 5% CO₂. After 18 hours of growth,the plates were washed with 2 mL DPBS and the resulting solution wasdiluted with additional DPBS and mixed 1:1 of culture and FBS(pre-warmed to RT) to obtain a final concentration of between 5×10⁸ and1×10⁹ CFU/dose. After the FBS was added, the cultures were incubated for5 min. at RT to up-regulate H. somni virulence factors, and then placedon ice.

Each group of ten, 18-20 g NSA: CF-1 mice (Harlan SpragueDawley)/isolate were injected with 0.5 cc intraperitoneally (IP) withits assigned isolate. A control group of ten mice was given DPBS+FBS.Filter-tops were placed on the top of each of the mice cages with onecage equaling ten mice/isolate. The isolates were diluted and plated,and plates were incubated at 37° C. with 5% CO₂ for two days. Plateswere counted and the actual CFU/dose was determined for each isolate.Additionally, five mice were injected SQ with isolates 2336 or TK #33,to determine if this route of administration would be a viablealternative (for assessing virulence) to the IP route. After challenge,mice were observed daily for mortality.

Most isolates yielded mid to upper 8 logs CFU/dose. The SQ method provedto be an unsuccessful method for challenging mice. Isolates TK #4, TK#24, TK #14, the controls, and 2336 resulted in the least percentmortality, while isolates TK #1, TK #2, TK #21, TK #28, and TK #30resulted in the greatest percent mortality (Table 2).

TABLE 2 Percent mortality when 18-20 g mice were challenged withplate-grown isolates. Isolate # Route CFU/dose % Mortality TK #33 SQ 3.7× 10⁸ 0 Controls IP 0 0 2336 IP 1.7 × 10⁹ 0 2336 SQ 1.7 × 10⁹ 0 TK #4 IP 7.6 × 10⁸ 10 TK #14 IP 8.7 × 10⁸ 10 TK #24 IP 5.5 × 10⁸ 10 TK #34 IP7.2 × 10⁸ 20 TK #42 IP 5.9 × 10⁸ 20 TK #5  IP 6.2 × 10⁸ 30 TK #15 IP 7.5× 10⁸ 30 TK #33 IP 3.7 × 10⁸ 30 TK #37 IP 7.3 × 10⁸ 30 TK #41 IP 9.9 ×10⁸ 30 TK #3  IP 8.6 × 10⁸ 40 TK #22 IP 8.0 × 10⁸ 40 TK #40 IP 9.9 × 10⁸40 TK #44 IP 9.0 × 10⁸ 40 TK #47 IP 7.0 × 10⁸ 40 TK #1  IP 8.0 × 10⁸ 50TK #2  IP 8.7 × 10⁸ 50 TK #28 IP 7.1 × 10⁸ 50 TK #30 IP 4.8 × 10⁸ 50 TK#21 IP 5.1 × 10⁸ 80

Challenge Model.

Two studies were conducted to determine the ideal CFU/dose and challengeculture preparation for mice that would be larger invaccination-challenge studies than in the previous study (i.e. takinginto account the three weeks' growth between vaccination and challenge).For the first study, forty, 25-30 g NSA: CF-1 mice (Harlan SpragueDawley) were used. H. somni Isolate TK #21 was streak-plated onto CSBA.After ˜24 hours of growth at 37° C. with 5% CO₂ the isolate was lawnedonto additional CSBAs and grown under the same conditions for 18-20hours. Each plate was washed with 2 mL DPBS and the liquid wascollected. Dilutions from the original plate wash were made to createtwo additional solutions varying in CFU/dose. Each dilution was mixed1:1 with FBS to achieve final concentrations of challenge fluids rangingfrom low 8 logs to low 9 logs/dose. Control fluids were created with 1:1DPBS and FBS. All FBS added cultures were incubated at room temp. for 5min. and then placed on ice. Mice were challenged with 0.5 cc IP andmonitored for mortality after challenge. The challenge cultures werediluted and plated to determine the ideal CFU/dose required for maximummortality.

When the challenge culture TK #21 was grown on plates and mice werelarger (25-30 g), to account for their expected size three-weekspost-vaccination, the greatest percent mortality was achieved at 5.7×10⁸CFU/dose (Table 3).

TABLE 3 Percent mortality when 25-30 g mice were challenged withplate-grown isolates. Group Isolate CFU/dose % Mortality 1 TK #21 1.6 ×10⁹ 9.1 2 TK #21 5.7 × 10⁸ 27.3 3 TK #21 4.2 × 10⁸ 20 4 Control 0 0

A final challenge study was conducted with thirty, 25-30 g NSA: CF-1mice (Harlan Sprague Dawley) to compare the difference between growingthe challenge culture on plates (as is commonly done in the literature)or in broth. Similar as above, isolate TK #21 was removed from frozenstorage and lawned onto additional plates. For the broth culture, aplate was washed with 2 mL Columbia broth and 50 μL was added to 25 mLpre-warmed Columbia broth. The culture was shaken vigorously after theculture wash was added and then placed on a shaker set to 37° C. and 250rpm. Growth was monitored and the culture was stopped when we estimatedthe final challenge culture, with all fluids added, would contain mid-8logs/dose. Also, a challenge culture was prepared by plate washing asabove, with the culture diluted with DPBS to about the sameconcentration as the broth culture. Both the broth and plate cultureswere mixed 1:1 with FBS and incubated for 5 min. at room temp. prior tobeing placed on ice. Eleven mice/method were challenged with 0.5 cc IPof the plate wash or the broth and 10 mice served as controls given amixture of equal parts Columbia broth and DPBS, which was then mixed 1:1with FBS. Mice were monitored for mortality post-challenge and dilutionsand plating was completed to determine CFU/dose.

Greater mortality of 25-30 g mice occurred when mice were challengedwith isolate #21 grown in broth compared to the same isolate grown onplates (Table 4). This result was unexpected, particularly consideringthe literature guided the skilled person away from growing H. somni inbroth for use in a virulent challenge study (Berghaus et. al., 2006;Geertsema et. al., 2008; Gershwin et. al., 2005).

TABLE 4 Percent mortality when 25-30 g mice were challenged withplate/broth-grown isolates. Method of Growth Isolate CFU/dose %Mortality Plates TK #21 5.3 × 10⁸ 36.4 Broth TK #21 4.8 × 10⁸ 54.5 —Controls — 0

One hundred thirty NSA: CF-1 mice, 18-20 g were purchased from HarlanSprague Dawley. Five H. somni isolates (TK #42, TK #14, TK #4, TK #24,and TK #34) were selected from the previous studies as vaccinecandidates. These isolates were removed from storage and streaked ontoCSBA where they were incubated for ˜24 hours at 37° C. and 5% CO₂. Theisolates were transferred to new CSBAs and lawned onto the plate surfacewhere they were allowed to grow for ˜18 hours. The plates were washedwith 2 mL Columbia broth. For isolates TK #42 and TK #34, 100 μL ofplate wash was added to 25 mL of Columbia broth, and for isolates TK #4,TK #14, and TK #24, 50 μL of plate wash was added to 25 mL Columbiabroth, based on previous studies involving the growth of these isolates.

The cultures were incubated at 37° C. and 200 rpm. Growth of theisolates was monitored and cultures were removed from incubation anddiluted with Columbia broth to contain approximately 1×10⁸ CFU/dose anda 1:10 dilution of this was made to make another vaccine estimated at1×10⁷ CFU/dose, for each isolate. A control group was vaccinated withsterile Columbia broth and another control group was vaccinated with thecommercially available SOMUBAC® (Zoetis, inactivated Histophilusvaccine, adjuvanted with aluminum hydroxide, Kalamazoo, Mich.).

Each vaccine was maintained on ice after it was prepared and each groupincluded 10 mice, with the exception of the Columbia Control group whichincluded 6 mice. The vaccine was delivered SQ as 0.5 cc. Aftervaccination, the vaccines were diluted and plated to determine actualCFU/dose and mice were monitored daily for adverse reactions and/ordeath. Endotoxin involved in each vaccine was determined with theLimulus Amebocyte Lysate kit (Lonza, Allendale, N.J.).

Nineteen days after vaccination, mice were challenged with H. somniisolate TK #21. The wildtype was prepared for growth in broth similar asabove, with the exception that 150 μL of plate wash was used toinoculate each of 2 flasks containing 75 mL pre-warmed Columbia brotheach. The flasks were incubated at 37° C. and 250 rpm. The culture wasremoved from incubation, diluted, and mixed 1:1 with room temperatureFBS to contain mid-8 logs/dose. The culture was incubated with the FBSat room temperature for 5 min. and placed on ice. The challenge productwas dilution plated prior-to- and post-challenge to determine if theCFU/dose matched our estimate. Mice were administered 0.5 cc IP of thechallenge culture and monitored daily for death.

Mice vaccinated with SOMUBAC® (Zoetis, inactivated Histophilus vaccine,adjuvanted with aluminum hydroxide), and isolates TK #34 and TK #22 at 8logs, developed red injection site reactions. All isolates, with theexception of TK #22, provided some protection to mice prior tochallenge, in comparison to the control group. Isolate TK #4 waseffective at both vaccine doses, however, isolate TK #34 was best at 8logs and isolates TK #24 and TK #42 were best at 7 logs. At both doses,isolate TK #14 provided some protection but less than the others (Table5). Isolates TK #24 at 8 logs and TK #42 at 8 logs had one mouse of 10die after vaccination, prior to challenge.

TABLE 5 The percent mortality in mice vaccinated with a low virulenceisolate and challenged with a high virulence isolate. Vaccine VaccineChallenge % Isolate CFU/Dose EU/dose* CFU/dose Mortality TK #4  1.8 ×10⁷ — 5.1 × 10⁸ 10 TK #4  1.8 × 10⁸ 31,559 5.1 × 10⁸ 20 TK #14 1.8 × 10⁷— 5.1 × 10⁸ 40 TK #14 1.8 × 10⁸ 30,287 5.1 × 10⁸ 40 TK #22 1.3 × 10⁷ —5.1 × 10⁸ 60 TK #22 1.3 × 10⁸ 54,451 5.1 × 10⁸ 80 TK #24 1.3 × 10⁷ — 5.1× 10⁸ 10 TK #24 1.3 × 10⁸ 38,372 5.1 × 10⁸ 33 TK #34 1.6 × 10⁷ — 5.1 ×10⁸ 40 TK #34 1.6 × 10⁸ 28,107 5.1 × 10⁸ 0 TK #42 1.3 × 10⁷ — 5.1 × 10⁸10 TK #42 1.3 × 10⁸ 33,648 5.1 × 10⁸ 33 SOMUBAC ® — — 5.1 × 10⁸ 10Control 0 2,307 5.1 × 10⁸ 67 *EU/dose = Endotoxin units/dose; EU/dosewas only determined for the 8 log vaccines.

Example 2—H. somni Calf Challenge

Prior to testing the vaccine candidates, a calf challenge model had tobe developed. For this study, twenty Holstein bull calves were used.Calves were challenged at 54 days-of-age using either isolate TK #21 or153-3 (acquired from calves with confirmed clinical disease due to H.somni). The challenge culture was prepared in broth similar to above. Toestimate the CFU/dose of the challenge culture, a previously establishedstandard curve (y=−5×10⁻⁸x+92.017 (x=CFU/mL and y=% T (540 nm)) was usedto determine the dilutions needed to achieve a final challengeconcentration of ˜2.5×10⁸ and 2.5×10⁹ CFU/dose for each isolate. Theculture was diluted with EBSS followed by the addition of a 1:1 ratio ofFBS after which the cultures were incubated for 5 min. at room temp. Thechallenge cultures were placed on ice along with additional EBSS forchasing the challenge dose. For each of 4 calves per isolate and dose,20 cc of challenge was administered trans-tracheally and was chased intothe lungs with 60 cc of EBSS. Four animals remained as non-challengedcontrols, which were given 1:1 EBSS and FBS. The challenge culture wasdiluted and plated prior-to- and post-challenge to determine actual CFUadministered. Clinical signs were rated and recorded daily by the sameperson as follows:

TABLE 6 Calf Challenge Clinical Signs Rating Criteria Rating CriteriaAttitude 0 Normal 1 Depressed 2 Moderately Depressed 3 SeverelyDepressed Anorexia 0 Normal 1 Little motivation to eat 2 No motivationto eat 3 No motivation to eat or interact → Humanely EuthanizeRespiration Rate 0 Normal 1 Increased Respiration Rate 2 LaboredBreathing 3 Labored Breathing and Wheezing Nasal Discharge 0 Normal 1Small amount of unilateral discharge 2 Bilateral, excessive mucusdischarge 3 Thick bilateral discharge Cough 0 Normal 1 Single Cough 2Occasional spontaneous cough or repeated coughs 3 Repeated spontaneouscoughs Ability to Rise or Walk 0 Normal 1 Rise with enticement andmotivated to walk 2 Rise with enticement and no motivation to walk 3 Nomotivation to rise/walk → Humanely Euthanize

The total clinical score was calculated for each animal by summing theratings determined for each category. If an animal was found dead, ormet the criteria of euthanasia, a necropsy was performed and the lungsremoved. An attending veterinarian rated the lungs for % surface arealesions, and tissues were subjected to analysis. After 4 days, theremaining animals were euthanized and necropsied as above. Lung lesionswere arcsine-transformed and analyzed using the “JMP Fit Y by X”function and means were separated with Student's t.

Twenty-four Holstein bull calves were divided into five pens containingfive animals each for vaccination and 4 animals to serve asnon-vaccinated controls. In addition, four animals from the challengemodel's non-challenged group were kept alive given the calfvaccination-challenge study was scheduled to be challenged the followingweek. They were kept in a separate pen. These animals, referred to ascontrols from challenge model, were an additional experimental controlto see if being in the same barn but not the same pen as challengedanimals could affect the results when these animals were challenged.Vaccines were prepared as for mice using isolates TK #28, TK #42, TK #4,and TK #34. The vaccine cultures were prepared similarly to the abovechallenge culture without the addition of FBS. The vaccines wereestimated to contain 5×10⁸ CFU/dose and a dose was 2 cc, 1 ccadministered in each calf nostril.

The vaccines were diluted and plated prior-to- and post-vaccination todetermine the actual CFU/dose. Animal health was monitoredpost-vaccination. Nasal swabs and blood samples were collected fromanimals on day −17 (one day after arrival), day 0 (date of vaccination,calf age=35 days-old), day 14, day 21, and day 33 (date of challenge).Nasal swabs were submitted to the Newport Laboratories Diagnostic Labfor bacterial culture and blood samples were maintained for possibleserology. All animals with the addition of the 4 control animals fromthe challenge model study, were challenged the same as above with anestimated 5×10⁹ CFU/dose. Clinical signs, necropsies, lung lesions,bacterial culture, Mycoplasma PCR, and statistics were carried out asabove with total clinical score and lung lesions also analyzed with JMPFit Y by X.

For the challenge study, the actual plated CFU/dose was very close tothe estimated 2.5×10⁹ or 2.5×10⁸ CFU/dose that was predicted. Theanimals challenged with isolate TK #21 were given 3.9×10⁹ CFU/dose forthe 9 log group and 2.9×10⁸ CFU/dose for the 8 log group. The animalschallenged with isolate 153-3 were given 1.8×10⁹ CFU/dose for the 9 loggroup and 1.7×10⁸ CFU/dose for the 8 log group.

More clinical signs of disease were observed for animals challenged withisolate TK #21 at 9 logs than any other challenge group (Table 7). Inaddition, more severe lung lesions were observed for animals challengedwith isolate TK #21 than any other challenge group (FIG. 1). Themajority of calves that had pneumonic lesions had H. somni recoveredfrom the lung tissue. Throughout the study, P. multocida was recoveredfrom the nasal swabs and the lung tissue, but did not appear to be acontributing cause of the disease symptoms, as animals did not showclinical signs of pneumonia until post-challenge. Also, many of theanimals were confirmed positive or suspect for M. bovis in the lungtissue. As expected, no M. bovoculi was detected.

TABLE 7 The average sum of clinical signs rated during the challengemodel study. Isolate Average Clinical Score* 153-3 (9 logs) 0.75 153-3(8 logs) 0.25 Controls 0.00 TK #21 (9 logs) 7.00 TK #21 (8 logs) 1.75*From the last rating taken of the animals prior to death.

The actual concentration of bacteria used for vaccination was close tothe estimated 5×10⁸ CFU/dose. H. somni was recovered only aftervaccination with the live bacteria from two to three animals at each ofthe nasal swab collection dates of day 14, day 21, and day 33post-vaccination. The nasal swabs demonstrated that H. somni can remainviable in the nasal passages for 21-33 days if not more (Table 8).

TABLE 8 Bacteria concentration and endotoxin administered to calvesvaccinated intranasally with isolates TK #28, TK #42, TK #4, and TK #34,and the number of H. somni isolates recovered from the vaccinatedanimals throughout the study. Vaccine Actual CFU/dose Endotoxin NasalSwab Recovery Isolate administered Units/mL (day) of recovery)* TK #283.90 × 10⁸ 27,800 2 calves (d21) 1 calf (d33) TK #42 4.40 × 10⁸ 31,700 1calf (d14) TK #4  7.80 × 10⁸ 25,800 1 calf (d21) TK #34 6.20 × 10⁸42,000 2 calves (d14); 1 calf (d33) Control 0 <10,000 None

The concentration of isolate TK #21 given to each calf at the time ofchallenge was 7.39×10⁹ CFU/dose. The challenge was effective indetermining the efficacy of the vaccines and to determine the mosteffective vaccine candidate. The clinical signs and percent of lunglesions show that isolate TK #4 was the most effective vaccine andisolate TK #34 was the least effective, while isolates TK #28 and TK #42fall in between (Table 9; FIG. 2). H. somni was recovered from the lungsof all calves in the study, with the exception of 3 calves all of whichwere vaccinated with isolate TK #4, indicating that cause of deathand/or disease for animals with lung lesions was attributable to thechallenge isolate. During the study P. multocida was recovered from thenasal passages of some of the calves, increasingly as the studyprogressed. After challenge, P. multocida was recovered from the lungtissue of 9 of the 28 calves and M. bovis was detected in the lungs fromthree of the animals. Vaccination does not appear to correlate with thepresence or absence of P. multocida or M. bovis. In addition, controlsfrom the challenge model were not as susceptible to the challenge whencompared to the control animals from this study. Since the challengemodel controls were housed in the same barn as challenged animals, theymay have acquired some immunity from previous exposure (Table 9).

TABLE 9 Clinical scores from animals vaccinated intranasally andchallenged with H. somni wildtypes. Isolate Average Clinical Score* TK#28  5.80 BC TK #42  6.00 BC TK #4  1.40 C TK #34 11.80 A Controls 10.50AB Controls from Challenge Model  3.50 C *Is the average clinical scoresfrom the last rating taken of the animals prior to death. Clinicalscores with different letters are significantly different according toStudent's t test.Discussion.

As the data in the mouse model correlated well with the results obtainedin calves, the inventors have provided a new and useful tool forselecting bovine vaccine/challenge candidates. Isolate TK #21 is anexcellent challenge isolate due to its virulence and the reproducibilityof the results. The ideal challenge dose appears to be around mid- tohigh-9 logs per dose in cattle when administered trans-tracheally.

When used as an intranasal vaccine, Isolate TK #4, was significantlyprotective against subsequent H. somni challenge (4 of 5 calves).Clinical signs and lung lesions were significantly different fromnon-vaccinates. Isolates TK #28 and TK #42 induced some protection invaccinated calves, but the efficacy was lower (2 of 5 calves).

Example 3—Comparative Genomic Analysis of Virulent and Avirulent H.somni Isolates

As discussed above, various H. somni isolates had different levels ofvirulence in mice and cattle. One in particular, TK #4, showed promiseas an intranasal cattle vaccine candidate (Example 2). In addition, TK#21 proved to be a highly virulent challenge isolate for cattle andmice, possessing a phenotype typical of challenge isolates used in theliterature (2336 and HS91). To better understand the factors that affectthis virulence and also to determine if recently acquired isolatesremain similar to older isolates, eight H. somni isolates were submittedfor whole genome sequencing. Four genes, a glycoside hydrolase familyprotein, a lipoprotein, a multicopper oxidase type 3, and a TetR familytranscriptional regulator were found to be missing in TK #4, but presentin TK #21. Also, the recently isolated H. somni lacked 211 to 316 genespresent in 2336, a wildtype isolated in 1985. HS91, an isolate from1991, lacked only 15 genes when compared to 2336. The data suggests thatthe genotype of TK #4 contributes to its avirulent phenotype, and thatthere is genetic drift within the H. somni population over time. Theresults demonstrate a genetic explanation for the attenuation of TK #4and the need for current vaccine and challenge isolates to maintainefficacy in response to natural genetic drift.

To understand the genetic and molecular basis for the differentialvirulence a 9-way comparative genome analysis was carried out between TK#4, TK #21, TK #34, TK #28, TK #42, HS91, 2336 (a highly virulentstrain, at least according to the literature), 129PT (a known avirulentstrain in GenBank), and 153-3 (a moderately virulent strain), againstGenBank isolate 2336. This analysis will also improve our understandingof possible genetic drift, and delineate genes involved in thevirulence/avirulence mechanism.

Material and Methods

Sample Preparation for Sequencing

Eight H. somni isolates were identified as a diverse set of isolatesthat could be valuable in whole genome sequencing (Table 10). Theseisolates were from a number of locations, had a broad range ofvirulence, and were isolated from animals in different years. All theseisolates were grown in Columbia broth (BD Ref#294420; Lot#0292713,Franklin Lakes, N.J.) and after growth, 10% glycerol was added to freezethe cultures in cryogenic vials (Fisher Scientific Cat.#10-500-26,Waltham, Mass.) at Newport Laboratories Research and Developmentfacility (Worthington, Minn.) in a −80° C. freezer. The isolates wereremoved from the freezer and streak-plated onto Columbia Sheep BloodAgar plates (CSBA) (BD Ref#22165/221263; Lot#2227132 2012 11 13,Franklin Lakes, N.J.). The plates were incubated overnight at 37° C.with 5% CO₂. After 24-26 hours, the plates were removed from theincubator and used to lawn additional CSBA for growth overnight at 37°C. and 5% CO₂. After 16-18 hours of incubation the plates were washedwith 2 mL pre-warmed Columbia broth with 100 μL or 150 μL used to startbroth cultures of 25 mL Columbia broth for each of the cultures, usingone flask for each inoculum amount. The broth cultures were shaken at200 rpm and percent transmittance (% T)(540 nm) was monitored until thecultures reached 15-20% T. The broth cultures were removed from theincubator, pelleted three times in the same microcentrifuge tube byspinning 1.5 mL of culture each time for 2 min. at 15,000 rpm, andgenomic DNA was isolated using a Bacterial Genomic DNA Purification Kit(Edge Biosystems, Gaithersburg, Md.), following the manufacturer'srecommendation with slight modification. DNA was extracted intriplicate, from the triplicate spin pellets of each culture. Amodification to the recommended DNA extraction process was the DNA wasresuspended in 100 μL of TE and it was incubated for 15 min. at 37° C.to promote dissolving the DNA pellet without shearing the DNA. The threeextractions were then pooled, compared on a 1% agarose gel, andquantified and purity checked on a Nanodrop. DNA amounts were calculatedin order to provide the required total DNA for sequencing at theUniversity of Idaho core facility (Moscow, Id.).

TABLE 10 List of H. somni isolates used for comparative genomicanalysis. Isolate Name Case # Location Phenotype TK #34 11-3452-2Fairmont, MN Reduced mortality in mice, but unable to reduce pneumoniaas a cattle vaccine HS91 NA Ames, IA Wildtype from 1991 TK #21 11-4134-1Marshall, MN Highly virulent to mice and calves in challenge studies TK#42 11-0141-1 Parkston, SD Had reduced virulence in mice and offeredsome protection as a mouse and cattle vaccine 2336 NA Dr. BriggsIndustry standard for virulent H. somni; however, we found challengeinconsistency making us question age and number of passages TK #2811-0125-1 Plainview, TX Offered some protection to cattle TK #412-0137-4 Grandview, ID Reduced virulence in mice, protected mice andcattle from challenge 153-3 NA Worthington, MN Wildtype recentlyobtained and seemed to be responsible for killing calves. In a calfchallenge titration study it was not as virulent as TK #21.Filtering High Quality Reads and Sequence Coverage

To compare 129PT from the NCBI database, Accession #CP000436, againstthe 2336 Accession #CP000947, a similar approach to the field isolatesneeded to be used. Therefore, pseudo-reads were generated from 129PTwith the ART read simulation software (hypertext transferprotocol://www.niehs.nih.gov/research/resources/software/biostatistics/art/).ART was run with the following command line: art_illumina -iHs129PT.fasta -p -l 250 -f 60 -m 500 -s 10 -na -o Hs129PT_sim, whichequates to -p=paired, -l=250 bp, -f=60x coverage, -m=mean size of DNAfragments, -s=10 standard deviation of DNA fragment size. Then, allisolate reads were filtered for quality with the Illumina TruSeqadapters using SeqyClean, which is a read cleaning program developed atthe University of Idaho (available on its website). This program removedthe sequencing adapters and low quality reads which contained 5 or morebases with a Q-value <20, thus eliminating error during base reads andincreasing accuracy.

Read Mapping

The genomic DNA results were mapped against 2336 in the NCBI database,Accession # CP000947 using bowtie2 default parameters. Filtering using acustom script for MAPQ <10 was used to remove multiply mapped reads. Itis expected that identical isolates have an alignment rate >95%.

Gene Coverage

Samtools was used to calculate mapping coverage for each positionagainst the 2336 reference. This was used to calculate the percentage ofbases that had coverage for each gene. Genes that contained less than80% of bases were called absent for that respective isolate. Twoanalyses were conducted on missing genes against 2336. The firstcompared HS91 (in a similar era as 2336), TK #21, TK #4, 153-3, and129PT. The second compared TK #34, TK #42, 2336 (to confirm our 2336matched the database), TK #28, and 129PT.

Pathogenicity Islands (PIs) and Integrative and Conjugative Elements(ICEs)

The results of the entire genome sequencing of HS91, TK #21, TK #4,153-3 and 129PT were compared to 2336. Pathogenicity islands (PIs) andIntegrative Conjugative Elements (ICEs) were searched for based on thepresence of flanking signature sequences like recombinases, integrases,transposases, helicases, and phage repeats. Genomic regions containing acluster of these putative signature sequences were identified aspotential PIs or ICEs.

Results

The cleaned sequences had an approximate coverage of 100× for allisolates. The extent of the coverage gives us confidence in the qualityof the called bases (Table 11).

TABLE 11 Quality of the raw reads and the estimated base coverage foreach sequenced isolate. Estimated Cleaned Bases Kept Estimated PESequenced Raw PE After Cleaned Library Reads Bases Coverage ReadsCleaning Coverage TK #34 454,197 235,274,046 103.93 460,095 210,850,73293.14 HS91 554,783 287,377,594 126.94 527,851 243,734,387 107.66 TK #21583,750 302,382,500 133.57 541,644 249,871,771 110.37 TK #42 527,632273,313,376 120.73 493,941 228,248,524 100.82 2336 848,512 439,529,216194.15 809,986 378,965,750 167.40 TK #28 544,568 282,086,244 124.60520,290 241,511,214 106.68 TK #4 606,064 313,941,152 138.68 570,358261,675,306 115.59 153-3 463,065 239,867,670 105.96 441,209 202,578,29289.48

The alignment revealed differences between the isolates and thereference strain. The GenBank 2336 isolate was 99.67% identical to the2336 genome sequenced by Newport Labs, indicating the validity andaccuracy of our map-based comparison between genomes. From therecomparisons on similarities of the other isolates to 2336 were made withthe second highest similarity to HS91. Also, the commensal 129PT(Accession # CP000436) in the NCBI database was aligned with thedatabase's 2336 as a reference comparison (Table 12).

TABLE 12 The percentage of the sequencing data that aligned with theGenBank 2336 Accession # CP000947. Isolates are listed from highest tolowest percent alignment rate. Isolate Alignment Rate (%) HS91 99.712336 99.67 153-3 96.11 TK #34 92.11 TK #4  91.30 TK #21 88.35 129PT*87.92 TK #42 83.84 TK #28 83.33 *Accession # CP000436.

Among the isolates, 129PT lacked many virulence and virulence-associatedgenes in comparison to the 2336 wildtype. The more recently acquiredisolates lacked more total genes than the older isolates, when comparedto the 2336 wildtype (Table 13).

TABLE 13 The number of genes estimated to be present or absent for eachisolate sequenced in comparison to 2336 Accession # CP00947 in the NCBIdatabase. Isolate Genes Present Genes Absent 129PT* 1593 472 TK #34 1749316 TK #4  1758 307 TK #21 1840 225 TK #42 1848 217 153-3 1851 214 TK#28 1854 211 2336 2048 17 HS91 2050 15 *Accession # CP000436.

When comparing the genes that are absent in the first and secondanalyses relative to 2336, 129PT, the commensal, lacked more of thosegenes associated with pathogenicity and/or virulence than any othercompared isolate. In total 129PT lacked 44 of the 47 total missing genescompiled from HS91, TK #21, TK #4, 153-3, and 129PT (Table 14). It alsolacked 44 of the 47 total missing genes compiled from TK #34, TK #42,2336, TK #28, and 129PT (Table 15). Isolate 2336 was found to lack onlyone identified pathogenicity-associated gene present in the NCBIdatabase. This gene is a YadA domain-containing protein (Table 15).

The vaccine candidate, TK #4 lacked a total of 23 of the possible 47absent genes from the isolates analyzed. This candidate lacked theattachment and adhesion genes of hemagglutinin/hemolysin, YadAdomain-containing proteins (a total of 5 repeats missing), and ahemagglutinin domain-containing protein. TK#4 also lacked a transferrinbinding protein (for iron uptake) and two isoforms of multicopperoxidase type 3 which provide the ability to uptake metals stored in thehost. Genes responsible for host colonization such as glycosidehydrolase family protein (nasopharnyx colonization) and peptidase S8/S53subtilisin kexin sedolisin were also missing from TK #4. Also, geneswere missing that are responsible for drug resistance or response tostressors including two isoforms of a TetR family transcriptionalregulator, acyltransferase 3, two isoforms of a MarR familytranscriptional regulator, small multidrug resistance protein, the MarRfamily transcriptional regulator, and a stress-sensitive restrictionsystem protein. Lastly, TK #4 lacked some genes that impact thepathogen's ability to evade host defenses such as lipooligosaccharidesialyltransferase, lipoprotein, and the Abi family protein (which isinvolved in self-immunity from bacteriocins) (Table 14).

In comparison to 2336, the highly pathogenic TK #21 also lacked similarvirulence genes found to be missing in TK #4 (19 of the 23 missing TK #4genes), with the exception of glycoside hydrolase family protein,lipoprotein, one isoform of multicopper oxidase type 3, and one isoformof the TetR family transcriptional regulator. The only gene that TK #21lacked that TK #4 had was one repeat of the YadA domain-containingprotein (Table 14).

HS91 was highly similar to 2336 and equally virulent according topublished literature. None of the virulence or virulence-associatedgenes that could potentially play a role in pathogenesis was found to bemissing in HS91 (Table 14).

Isolate 153-3 lacked many of the potential virulence genes found to bemissing in TK#4 (14 of the 23 missing in TK #4), but there were someadditional genes missing that were present in TK #4 (23 missing out of atotal 47 identified across all isolates). In addition to TK #4, 153-3lacked filamentous hemagglutinin outer membrane protein, adhesin, 10repeats of the YadA domain-containing protein (compared to 5 with TK#4), glycosyl transferase family protein, and acetyltransferase. Genesthat TK #4 lacked but were present in 153-3 were stress-sensitiverestriction system protein, hemagglutinin domain-containing protein,glycoside hydrolase family protein, lipooligosaccharidesialyltransferase, Abi family protein, lipoprotein, one isoform of themulticopper oxidase type 3, one isoform of the TetR familytranscriptional regulator, and peptidase S8/S53 subtilisin kexinsedolisin (Table 14).

The three recent isolates from the second analysis had more missinggenes than older isolates. TK #34 lacked the most genes of the recentisolates, 22 out of 47. These genes included filamentous hemagglutininouter membrane protein, hemagglutinin/hemolysin-like protein, adhesin,stress-sensitive restriction system, 6 repeats of the YadAdomain-containing protein, transferrin binding protein, two isoforms ofa hemagglutinin domain-containing protein, glycoside hydrolase familyprotein, TetR family transcriptional regulator, lob1 protein, twoisoforms of the MerR family transcriptional regulator, multicopperoxidase type 3, small multidrug resistance protein, MarR familytranscriptional regulator, and peptidase S8/S53 subtilisin kexinsedolisin. Of the 22 genes missing in TK #34, TK #42 was found to lack12. These included: hemagglutinin/hemolysin-like protein, 5 repeats ofthe YadA domain-containing protein, one isoform of the TetR familytranscriptional regulator, two isoforms of the MerR familytranscriptional regulator, one isoform of multicopper oxidase type 3,small multidrug resistance protein, and MarR family transcriptionalregulator. In addition to the 12 genes also absent with TK #34, TK #42also lacked one repeat of the YadA domain-containing protein, glycosyltransferase family protein, virulence associated protein D (VapD),acyltransferase 3, lipoprotein, another isoform of multicopper oxidasetype 3, and another isoform of the TetR transcriptional regulator. Allthe genes absent in TK #42 were also absent in TK #28, with noexceptions (Table 15).

TABLE 14 Genes involved in virulence that are missing in one or more ofthe analyzed genomes of HS91, TK #21, TK #4, 153-3, and 129PT, whencompared to 2336 (Accession # CP00947). NCBI Missing Gene # from? GeneName Gene Function HSM_0052 129PT Outer membrane Can involve porinsinvolved in transport of protein transport immunogenically importantproteins and host protein P1 evasion HSM_0077 153-3 & YadA domain-Promotes pathogenicity and virulence in host cells 129PT containingprotein through cell adhesion via a collagen binding outer-membraneprotein HSM_0164 129PT Glycosyl transferase Involved in transferring asugar moiety onto an family protein acceptor, responsible for generatingimportant virulence factors such as lipooligosaccharides (Kahler et. al.1996) HSM_0268 153-3 & Filamentous Mediates adherence to epithelialcells, 129PT hemagglutinin macrophages and is required for trachealouter membrane colonization protein HSM_0270 TK #21, Hemagglutinin/Surface glycoprotein responsible for bacterial TK #4, hemolysin-likeattachment to and penetration of host cells 153-3, & protein 129PTHSM_0274 153-3 & Adhesin Involved in cell attachment 129PT HSM_0338 TK#21, YadA domain- Promotes pathogenicity and virulence in host cells TK#4, containing protein through cell adhesion via a collagen bindingouter- 153-3, & membrane protein 129PT HSM_0346 153-3 & YadA domain-Promotes pathogenicity and virulence in host cells 129PT containingprotein through cell adhesion via a collagen binding outer- membraneprotein HSM_0377 TK #21, YadA domain- Promotes pathogenicity andvirulence in host cells TK #4, containing protein through cell adhesionvia a collagen binding outer- 153-3, & membrane protein 129PT HSM_0394153-3 & YadA domain- Promotes pathogenicity and virulence in host cells129PT containing protein through cell adhesion via a collagen bindingouter- membrane protein HSM_0598 TK #21, Stress-sensitive Under stressit can allow bacteria to more easily TK #4, restriction system pick upforeign DNA, such as phage DNA and protein (Schäferet. al. 1994) 129PTHSM_0695 129PT Alkaline Promotes biofilm formation phosphatase HSM_0708TK #21, YadA domain- Promotes pathogenicity and virulence in host cellsTK #4, containing protein through cell adhesion via a collagen bindingouter- 153-3, & membrane protein 129PT HSM_0749 TK #21, transferrinbinding Makes it possible for the bacteria to acquire stored TK #4 &protein iron from the host, which can affect virulence 153-3 HSM_0844129PT YadA domain- Promotes pathogenicity and virulence in host cellscontaining protein through cell adhesion via a collagen binding outer-membrane protein HSM_0953 TK #21 & Hemagglutinin Involved in bacterialadhesion TK #4 domain-containing protein HSM_0975 129PT GlycosylInvolved in transferring a sugar moiety onto an transferase familyacceptor, responsible for generating important protein virulence factorssuch as lipooligosaccharides (Kahler et. al. 1996) HSM_0977 153-3 &Glycosyl transferase Involved in transferring a sugar moiety onto an129PT family protein acceptor, responsible for generating importantvirulence factors such as lipooligosaccharides (Kahler et. al. 1996)HSM_0978 129PT Glycosyl transferase Involved in transferring a sugarmoiety onto an family protein acceptor, responsible for generatingimportant virulence factors such as lipooligosaccharides (Kahler et. al.1996) HSM_1089 129PT Hemolysin Involved in the lysis of red blood cellsactivation/secretion protein-like protein HSM_1090 129PT Filamentous Canbe involved in the secretion of adhesins that hemagglutinin facilitateadhesion to the host or other cells, outer membrane necessary for hostcolonization protein HSM_1160 TK #4 & Glycoside Can play a role inpeptidoglycan degradation and 129PT hydrolase family also incolonization of the nasopharnyx and protein invasion of host epithelialcells HSM_1191 TK #21, TetR family Involved in the transcriptionalcontrol of multidrug TK #4, transcriptional efflux pumps, pathways forthe biosynthesis of 153-3, & regulator antibiotics, response to osmoticstress and toxic 129PT chemicals, control of catabolic pathways,differentiation processes, and pathogenicity (Ramos et. al. 2005)HSM_1212 129PT hemagglutinin Involved in bacterial adhesiondomain-containing protein HSM_1257 TK #21, YadA domain- Promotespathogenicity and virulence in host cells TK #4, containing proteinthrough cell adhesion via a collagen binding outer- 153-3, & membraneprotein 129PT HSM_1426 TK #21, Lipooligosaccharide Affects theornamentation on the TK #4, & sialyltransferase lipooligosaccharide andimpacts the pathogen's 129PT ability to evade host defenses HSM_1448129PT Virulence- Toxin/antitoxin locus, can be involved in associatedprotein D translation during stressful conditions, translation (VapD)region arrest would improve survival in the host and promote chronicmucosal infections (Daines et. al. 2004) HSM_1484 153-3 & YadA domain-Promotes pathogenicity and virulence in host cells 129PT containingprotein through cell adhesion via a collagen binding outer- membraneprotein HSM_1542 153-3 & YadA domain- Promotes pathogenicity andvirulence in host cells 129PT containing protein through cell adhesionvia a collagen binding outer- membrane protein HSM_1571 TK #21 & YadAdomain- Promotes pathogenicity and virulence in host cells 129PTcontaining protein through cell adhesion via a collagen binding outer-membrane protein HSM_1616 TK #21, Abi family protein Involved inself-immunity to bacteriocins (Kjos et. TK #4, & al. 2010) 129PTHSM_1624 TK #21, Acyltransferase 3 Drug resistance and other hostevasion reponses TK #4, can be dependent on the presence of 153-3 &acyltransferases 129PT HSM_1647 129PT FhaB protein Can have a role inhost-cell binding and infection HSM_1651 129PT Filamentous Can beinvolved in the secretion of adhesins that hemagglutinin outerfacilitate adhesion to the host or other cells, membrane proteinnecessary for host colonization HSM_1667 129PT Lob1 protein Involved inlipooligosaccharide biosynthesis for evading or delaying hostrecognition (Cox et. al. 1998) HSM_1669 153-3 Acetyltransferase Involvedin evading the host immune system, virulence, and drug resistance (Aubryet. al. 2011) HSM_1714 TK #4 & Lipoprotein Expressed on the cell surfaceand can be involved 129PT in host evasion and virulence HSM_1726 TK #4 &Multicopper oxidase Could be involved in Iron (II) acquisition in low129PT type 3 oxygen environments from inflamed tissue, such as lungcolonization of the host (Huston et. al. 2002) HSM_1728 TK #21, MerRfamily Transcriptional regulator via a suboptimal TK #4, transcriptionalpromoter that responds to stressors such as 153-3, & regulator oxidativestress, heavy metals, or antibiotics 129PT (Brown et. al. 2003) HSM_1730TK #21, Multicopper oxidase Could be involved in Iron (II) acquisitionin low TK #4, type 3 oxygen environments from inflamed tissue, such153-3, as lung colonization of the host (Huston et. al. 129PT 2002)HSM_1734 TK #4, TetR family Involved in the transcriptional control ofmultidrug 129PT transcriptional efflux pumps, pathways for thebiosynthesis of regulator antibiotics, response to osmotic stress andtoxic chemicals, control of catabolic pathways, differentiationprocesses, and pathogenicity (Ramos et. al. 2005) HSM_1736 TK #21, Smallmultidrug Drug efflux protein involved in antibiotic TK #4, resistanceprotein resistance (Alekshun and Levy, 2007). 153-3 & 129PT HSM_1737 TK#21, MarR family Regulate activity of genes involved in antibiotic TK#4, transcriptional resistance, stress responses, virulence or 153-3 &regulator catabolism of aromatic compounds (Perera and 129PT Grove,2010) HSM_1741 TK #21, MerR family Transcriptional regulator via asuboptimal TK #4, transcriptional promoter that responds to stressorssuch as 153-3 & regulator oxidative stress, heavy metals, or antibiotics129PT (Brown et. al. 2003) HSM_1793 TK #21, YadA domain- Promotespathogenicity and virulence in host cells TK #4, containing proteinthrough cell adhesion via a collagen binding outer- 153-3 & membraneprotein 129PT HSM_1794 129PT Glycosyl transferase Involved intransferring a sugar moiety onto an family protein acceptor, responsiblefor generating important virulence factors such as lipooligosaccharides(Kahler et. al. 1996) HSM_1889 TK #21, Peptidase S8/S53 Functions as ahydrolase, protease, and serine TK #4, & subtilisin kexin protease thathelp with initial colonization of the 129PT sedolisin host

TABLE 15 Genes involved in virulence that are missing in one or more ofthe analyzed genomes of TK #34, TK #42, 2336, TK #28, and 129PT, whencompared to 2336 (Accession # CP00947 in the NCBI database). 2336 NCBIMissing Gene # from? Gene Name Gene Function HSM_0052 129PT Outermembrane Can involve porins involved in transport of protein transportimmunogenically important proteins and host protein P1 evasion HSM_0077TK #42, YadA domain- Promotes pathogenicity and virulence in host TK#28, & containing protein cells through cell adhesion via a collagen129PT binding outer-membrane protein HSM_0164 129PT Glycosyl Involved intransferring a sugar moiety onto transferase family an acceptor,responsible for generating protein important virulence factors such aslipooligosaccharides (Kahler et. al. 1996) HSM_0268 TK #34 & FilamentousMediates adherence to epithelial cells, 129PT hemagglutinin macrophagesand is required for tracheal outer membrane colonization proteinHSM_0270 TK #34, Hemagglutinin/ Surface glycoprotein responsible forbacterial TK #42, hemolysin-like attachment to, and penetration of, hostcells TK #28, & protein 129PT HSM_0274 TK #34 & Adhesin Involved in cellattachment 129PT HSM_0338 129PT YadA domain- Promotes pathogenicity andvirulence in host containing protein cells through cell adhesion via acollagen binding outer-membrane protein HSM_0346 129PT YadA domain-Promotes pathogenicity and virulence in host containing protein cellsthrough cell adhesion via a collagen binding outer-membrane proteinHSM_0377 129PT YadA domain- Promotes pathogenicity and virulence in hostcontaining protein cells through cell adhesion via a collagen bindingouter-membrane protein HSM_0394 129PT YadA domain- Promotespathogenicity and virulence in host containing protein cells throughcell adhesion via a collagen binding outer-membrane protein HSM_0598 TK#34 & Stress-sensitive Under stress it can allow bacteria to more 129PTrestriction system easily pick up foreign DNA, such as phage protein DNA(Schäfer et. al. 1994) HSM_0695 129PT Alkaline Promotes biofilmformation phosphatase HSM_0708 TK #34, YadA domain- Promotespathogenicity and virulence in host TK #42, containing protein cellsthrough cell adhesion via a collagen TK #28, & binding outer-membraneprotein 129PT HSM_0749 TK #34 Transferrin Makes it possible for thebacteria to acquire binding protein stored iron from the host, which canaffect virulence HSM_0844 129PT YadA domain- Promotes pathogenicity andvirulence in host containing protein cells through cell adhesion via acollagen binding outer-membrane protein HSM_0938 TK #34 HemagglutininInvolved in bacterial adhesion domain-containing protein HSM_0975 TK#42, Glycosyl Involved in transferring a sugar moiety onto TK #28, &transferase family an acceptor, responsible for generating 129PT proteinimportant virulence factors such as lipooligosaccharides (Kahler et. al.1996) HSM_0977 129PT Glycosyl Involved in transferring a sugar moietyonto transferase family an acceptor, responsible for generating proteinimportant virulence factors such as lipooligosaccharides (Kahler et. al.1996) HSM_0978 129PT Glycosyl Involved in transferring a sugar moietyonto transferase family an acceptor, responsible for generating proteinimportant virulence factors such as lipooligosaccharides (Kahler et. al.1996) HSM_1022 TK #34 YadA domain- Promotes pathogenicity and virulencein host containing protein cells through cell adhesion via a collagenbinding outer-membrane protein HSM_1089 129PT Hemolysin Involved in thelysis of red blood cells activation/secretion protein-like proteinHSM_1090 129PT Filamentous Mediates adherence to epithelial cells,hemagglutinin macrophages and is required for tracheal outer membranecolonization protein HSM_1160 TK #34 & Glycoside Can play a role inpeptidoglycan degradation 129PT hydrolase family and also incolonization of the nasopharnyx protein and invasion of host epithelialcells HSM_1191 TK #34, TetR family Involved in the transcriptionalcontrol of TK #42, transcriptional multidrug efflux pumps, pathways forthe TK #28, & regulator biosynthesis of antibiotics, response to 129PTosmotic stress and toxic chemicals, control of catabolic pathways,differentiation processes, and pathogenicity (Ramos et. al. 2005)HSM_1212 TK #34 & Hemagglutinin Involved in bacterial adhesion 129PTdomain-containing protein HSM_1257 TK #34, YadA domain- Promotespathogenicity and virulence in host TK #42, containing protein cellsthrough cell adhesion via a collagen TK #28, & binding outer-membraneprotein 129PT HSM_1426 129PT Lipooligosaccharide Affects theornamentation on the sialyltransferase lipooligosaccharide and impactsthe pathogen's ability to evade host defenses HSM_1448 TK #42,Virulence- Toxin/antitoxin locus, can be involved in TK #28, &associated protein translation during stressful conditions, 129PT D(VapD) region translation arrest would improve survival in the host andpromote chronic mucosal infections (Daines et. al. 2004) HSM_1484 129PTYadA domain- Promotes pathogenicity and virulence in host containingprotein cells through cell adhesion via a collagen bindingouter-membrane protein HSM_1542 TK #34, YadA domain- Promotespathogenicity and virulence in host TK #42, containing protein cellsthrough cell adhesion via a collagen TK #28, & binding outer-membraneprotein 129PT HSM_1571 TK #34, YadA domain- Promotes pathogenicity andvirulence in host TK #42, containing protein cells through cell adhesionvia a collagen TK #28, & binding outer-membrane protein 129PT HSM_1616129PT Abi family protein Involved in self-immunity to bacteriocins (Kjoset. al. 2010) HSM_1624 TK #42, Acyltransferase 3 Drug resistance andother host evasion TK #28, & responses can be dependent on the presence129PT of acyltransferases HSM_1647 129PT FhaB protein Can have a role inhost-cell binding and infection HSM_1651 129PT Filamentous Can beinvolved in the secretion of adhesins hemagglutinin that facilitateadhesion to the host or other outer membrane cells, necessary for hostcolonization protein HSM_1667 TK #34 & Lob1 protein Involved inlipooligosaccharide biosynthesis 129PT for evading or delaying hostrecognition (Cox et. al. 1998) HSM_1714 TK #42, Lipoprotein Expressed onthe cell surface and can be TK #28, & involved in host evasion andvirulence 129PT HSM_1726 TK #42, Multicopper Could be involved in Iron(II) acquisition in TK #28, & oxidase type 3 low oxygen environmentsfrom inflamed 129PT tissue, such as lung colonization of the host(Huston et. al. 2002) HSM_1728 TK #34, MerR family Transcriptionalregulator via a suboptimal TK #42, transcriptional promoter thatresponds to stressors such as TK #28, & regulator oxidative stress,heavy metals, or antibiotics 129PT (Brown et. al. 2003) HSM_1730 TK #34,Multicopper Could be involved in Iron (II) acquisition in TK #42,oxidase type 3 low oxygen environments from inflamed TK #28, & tissue,such as lung colonization of the host 129PT (Huston et. al. 2002)HSM_1734 TK #42, TetR family Involved in the transcriptional control ofTK #28, & transcriptional multidrug efflux pumps, pathways for the 129PTregulator biosynthesis of antibiotics, response to osmotic stress andtoxic chemicals, control of catabolic pathways, differentiationprocesses, and pathogenicity (Ramos et. al. 2005) HSM_1736 TK #34, Smallmultidrug Drug efflux protein involved in antibiotic TK #42, resistanceprotein resistance (Alekshun and Levy, 2007) TK #28, & 129PT HSM_1737 TK#34, MarR family Regulate activity of genes involved in TK #42,transcriptional antibiotic resistance, stress responses, TK #28, &regulator virulence or catabolism of aromatic 129PT compounds (Pereraand Grove, 2010) HSM_1741 TK #34, MerR family Transcriptional regulatorvia a suboptimal TK #42, transcriptional promoter that responds tostressors such as TK #28, & regulator oxidative stress, heavy metals, orantibiotics 129PT (Brown et. al. 2003) HSM_1793 TK #34, YadA domain-Promotes pathogenicity and virulence in host TK #42, containing proteincells through cell adhesion via a collagen 2336, outer-membrane proteinTK #28, & 129PT HSM_1794 129PT Glycosyl Involved in transferring a sugarmoiety onto transferase family an acceptor, responsible for generatingprotein important virulence factors such as ipooligosaccharides (Kahleret. al. 1996) HSM_1889 TK #34, & Peptidase S8/S53 Functions as ahydrolase, protease, and serine 129PT subtilisin kexin protease thathelps with initial colonization of edolisin the hostSeven Putative PIs or ICEs were Identified from the Whole GenomeCompilation.

The first one is located between HSM_R0009 to approximately HSM_0254(˜28 kb), but no notable virulence, virulence-associated, or drugresistance genes appear to be present in this location. HS91 and 2336contained all of the genes present; however, TK #21, TK #4, 153-3,129PT, and TK #34 appear to lack the majority of the genes located inthis PI or ICE. TK #42 and TK #28 have part of this first PI or ICE.

The second location was between HSM_0319 to HSM_0348 (˜41 kb), and atthis location there were two repeats of YadA domain-containing proteins,one which was lacking in TK #21, TK #4, 153-3, and 129PT and the otherwas missing in 153-3 and 129PT.

A third PI was located between HSM_0638 to HSM_0692 (˜45 kb). Isolates153-3, 129PT, and TK #34 lacked many of the genes in this potential PIor ICE; however, none were identified as putative virulence,virulence-associated, or drug resistance genes, because many of thegenes in the PI were only identified as hypothetical proteins.

The fourth PI location was between HSM_0847 and HSM_0923 (˜73 kb). Againno virulence factors were identified within this range; however TK #21,TK #4, 153-3, 129PT, and TK #34 lacked a majority of the genes found inthis region.

The fifth PI location was between HSM_1115 and HSM_1167 (˜51 kb). TK #4lacks HSM_1129 to HSM_1146 and HSM_1149 to HSM_1167, this absent regionincludes a glycoside hydrolase family protein. Isolate 129PT lacksHSM_1131 to HSM_1167, and TK #34 lacks HSM_1129 to HSM_1144 and HSM_1149to HSM_1167, but the other isolates have most of these genes present.There is also a two-component response regulator at HSM_1124 that can beinvolved in helping the bacteria sense and respond to a wide variety ofenvironments. All of the sequenced isolates contain this two-componentresponse regulator.

The sixth putative PI or ICE is from HSM_1615 to HSM_1719 (˜101 kb). Inthis region, there is an Abi family protein, acyltransferase 3, FhaBprotein, filamentous hemagglutinin outer membrane protein, Lob1 protein,acetyltransferase, and a lipoprotein. TK #21, TK #4, 153-3, 129PT, TK#28, TK #42, and TK #34 lack some of these (Tables 14 & 15), potentiallycontributing to their different levels of virulence.

The last potential PI or ICE is located at HSM_1860 to HSM_R0065 (˜38kb). One key gene that is missing from TK #21, TK #4, 129PT, and TK #34in this region is the peptidase S8/S53 subtilisin kexin sedolisin, whichis important for initial host colonization. There are other genesmissing from TK #21, TK #4, 129PT, TK #34, TK #42, and TK #28, most ofwhich are identified as hypothetical proteins or transposases. HS91,153-3, TK #42, 2336, and TK #28 appear to have most or all this entireregion intact.

Discussion

TK #4 appears to be sufficiently attenuated, while still capable ofeliciting an adequate immune response, making the strain a strongvaccine candidate. TK #4 lacks some genes similar to other isolatesacquired about the same year; however, some of the absent genes areunique to TK #4, including: a glycoside hydrolase family protein (GHFP),a lipoprotein (LP), a multicopper oxidase type 3, and a TetR familytranscriptional regulator. Several genes, missing from both TK #4 andpathogenic isolates, may contribute to the attenuated phenotype,however, these genes do not appear to be necessary for avirulence.

On the other hand, two genes uniquely absent from TK #4, while presentin pathogenic strains, glycoside hydrolase family protein andlipoprotein, appear to be necessary and sufficient for TK #4'sattenuated phenotype.

A causal link between the absence of expression of these two genes—in TK#4—and the attenuated virulence is supported by previously publishedwork, which indicates the GHFP and LP proteins play an important role incolonization and evasion of host defenses (Asgarali et. al. 2009; Garbeand Collin, 2010; Liu et. al. 2008; and Guzmán-Brambila et. al. 2012).Moreover, the data indicate that while TK #4 is not as attenuated as129PT, the strain affords protection (from subsequent virulentchallenge) to both cattle and mice.

TK #34 is also highly attenuated similar to TK #4; however, TK #4 ismissing a greater number of genes involved in host evasion. TK #4 and TK#34 both lack various isoforms or repeats of hemagglutinindomain-containing proteins and YadA domain-containing proteins,respectively. TK #34 lacks a filamentous hemagglutinin outer membraneprotein, adhesion, and the lob1 protein, which TK #4 has. TK #4 lacks alipooligosaccharide sialyltransferase, Abi family protein,acyltransferase 3, lipoprotein, multicopper 3, and TetR transcriptionalregulator that are present in TK #34. The results indicate that thecombination of missing virulence factors contributes to a partiallyattenuated bacteria that is protective in mice and cattle (TK #4) or toa highly attenuated but not protective isolate for mice and cattle (TK#34). The data here alone indicate that a skilled person could not havepredicted ahead of time which genes should be deleted to obtain asufficiently attenuated, yet sufficiently protective, H. somni vaccinestrain.

Finally, horizontal gene transfer appears not to have passed someresistance genes to this isolate, and a lack of other virulence factorsappear to have attenuated the isolate sufficiently to render itincapable of causing disease. On the other hand, the presence of morevirulence factors than a commensal strain makes TK #4 a strong vaccinecandidate (i.e. the strain can survive long enough to stimulate the hosthumoral immune response and provide longer-term protection).

TK #21 was found to be a highly virulent isolate in mice and cattle.However, genetically, it was more similar to current isolates, TK #4 and153-3 (less virulence relative to TK #21), while at the same time moredivergent from other known virulent strains, 2336 and HS91. TK #21 didpossess a, glycoside hydrolase family protein, a lipoprotein, oneisoform of multicopper oxidase type 3, and one isoform of the TetRfamily transcriptional regulator that TK #4 lacked. These 4 genes mayall contribute to the attenuation of TK #4. The most unique genesmissing that were not found missing as different isoforms in TK #21,were glycoside hydrolase and a lipoprotein. These genes in combinationappear to be necessary for causing the sufficient difference between achallenge isolate (has the genes) and a vaccine candidate (lacks thegenes). TK #21 did lack a Yad A domain-containing protein that TK #4had; however, it does not seem to contribute to lack of virulence. Itseems that virulence associated with TK #21 is not due to its divergencefrom 2336, but instead to its subtle differences from TK #4.

The more recent isolates of TK #21, TK #4, 153-3, TK #34, TK #42, and TK#28 appear to be similar, but they are notably different from 2336 andHS91 (see Table 15). The data suggests that the H. somni population isevolving over time and that the generally accepted challenge isolate of2336 may no longer be relevant to the H. somni gene pool calves arecurrently facing. This also provides evidence to support TK #4 as avaccine candidate, since its genetic composition is highly relevant tocurrently circulated strains in the calf bovine respiratory diseasecomplex. The alignment rate supports that HS91 and 2336 are very closelyrelated, and that TK #21, TK #4, 153-3, TK #34, TK #42, and TK #28 areclosely related. It also shows that the current isolates are geneticallydifferent from those isolated in the 1980-1990s. Also, when looking atgene absence, the more current isolates lack higher numbers of 2336genes than HS91, and the current isolates also tend to lack similargenes. This implies that genomes are naturally drifting in H. somni.Also, since the current isolates represent different states within theU.S., it implies that the genetic drift seen here is representative ofthe current H. somni population in the U.S.

Overall, this data indicates that a combination of a number of genes maybe required to make an isolate an effective vaccine or challengecandidate, and the loss of only a few genes can attenuate the candidateenough to save an animal versus take its life. This data also suggests,that the H. somni population does drift over time and that the industryneeds to evolve in order to maintain relevant vaccines. The datastrongly supports the importance of autogenous vaccines, as well as,periodic re-evaluation of commercial vaccine efficacy.

TABLE 16 The variants identified (with respect to 2336, accession #CP00947) for each isolate analyzed with “SAMTools mpileup.” Sample TotalVariants Filtered variants 129PT 22,040 21,733 TK #34 23,386 23,179 HS91132 12 HS91 ΔaroC 1,481 1,343 TK #21 25,840 25,157 TK #42 21,833 21,6772336 (Applicants' isolate) 134 15 TK #28 21,803 21,590 TK #4 25,86825,176 153-3 21,260 20,750 HS91 ΔnanPU 1,468 1,330

In addition to many SNPs identified (Table 16), there were also numerousmissing genes (Tables 17-23). The more recent isolates (TK #21, TK #4,153-3, TK #34, TK #42, and TK #28) and 129PT had the most SNPs, whilethe HS91 wildtype, HS91 mutants, and 2336 had few to no SNPs for allvirulence-associated genes. For the LOS biosynthesis or modificationgenes, the highest percentage SNPs were found in the glycosyltransferase and acetyltransferase genes in both analyses, and in lob2bof the group 2 analysis. Isolate 129PT lacked the most LOS biosynthesisor modification genes. For the adhesion, colonization, and biofilmformation genes, the most SNPs occurred in the YadA domain-containingproteins and the hemagglutinin domain-containing proteins. Many of theadhesion, colonization, and biofilm formation genes were missing in therecent isolates. Glycoside hydrolase in group 1 and in both groups someof the TonB-dependent receptors had the most SNPs for host invasion ormetal uptake. Many of the stress-response, antibiotic resistance genes,and drug efflux genes were missing from most of the newer isolates. Ofthose that were present, the percentage of SNPs was fairly low with theexception of the TetR family transcriptional regulator in group 1.

For genes involved in LOS biosynthesis or modification, all isolateswith the exception of 129PT had 1 indel in OMP transport protein P1. TK#21 and TK #34 had 1 and 2 indels, respectively, for lob2b. All recentisolates had 1 indel for pgmB, but the older isolates of HS91 and 2336did not. TK #42 and TK #28 had 1 indel in a glycosyl transferase familyprotein, and they had 1 indel in lob2a. Isolate 129PT had an indel inneuA_(HS).

In the group 1 analysis of non-synonymous indels forvirulence-associated genes involved in adhesion, colonization, andbiofilm formation, TK #21 and TK #4 had the most indels. These occurredin the YadA domain-containing proteins (26 total indels for TK #21 and32 total indels for TK #4), filamentous hemagglutinin outer membraneproteins (6 total indels for TK #21 and 5 total indels for TK #4), and 1indel for TK #21 in an adhesin. Isolate 153-3 also had some indels ingroup 1 but lacked the majority of the genes analyzed, somewhat similarto 129PT. Of those genes present with non-synonymous indels were YadAdomain containing proteins (total of 6 indels), a filamentoushemagglutinin outer membrane protein (3 indels), and a hemagglutinindomain-containing protein (1 indel; Table 19). For the analysis on group2 of the adhesion, colonization, and biofilm formation, isolates TK #34,TK #42, and TK #28 had the most indels. The indels were present in YadAdomain-containing proteins (24 total indels for TK #34, 31 total indelsfor TK #42, and 31 total indels for TK #28), filamentous hemagglutininouter membrane proteins (2 total indels for TK #34, 4 total indels forTK #42, and 4 total indels for TK #28) and 1 indel for a hemagglutinindomain-containing protein for TK #34. Also, 129PT had 7 indels for theYadA domain-containing proteins and 1 indel in a hemagglutinindomain-containing protein, although it lacked many of the genesanalyzed. Lastly, HS91 ΔaroC and HS91 ΔnanPU had 3 and 2 total indels,respectively, for a filamentous hemagglutinin outer membrane protein and1 each for a YadA domain-containing protein (Table 20).

TABLE 17 Number of indels present in isolates from group 1 analysis ofvirulence-associated genes involved in lipooligosaccharide (LOS)biosynthesis or modification. # of Non-synonymous Indels Present GeneNumber Total bp HS91 TK #21 TK #4 153-3 129PT HSM_0052 234 1 1 1 1 *HSM_0164 1569 0 0 0 0 * HSM_0204 1383 0 0 0 0 0 HSM_0397 930 0 0 0 0 0HSM_0572 555 0 0 0 0 0 HSM_0840 588 0 0 0 0 0 HSM_0925 1431 0 0 0 0 0HSM_0938 1167 0 0 0 0 0 HSM_0975 696 0 0 0 0 * HSM_0977 834 0 1 0 * *HSM_0978 816 0 0 0 0 * HSM_1062 1365 0 0 0 0 0 HSM_1063 888 0 0 0 0 0HSM_1117 672 0 0 0 0 1 HSM_1118 1020 0 0 0 0 0 HSM_1256 1017 0 0 0 0 0HSM_1426 903 0 * * 0 * HSM_1667 129 0 0 0 0 * HSM_1669 876 0 0 0 * 0HSM_1714 630 0 0 * 0 * HSM_1794 936 0 0 0 0 * HSM_1832 1656 0 1 1 1 1 *Indicates when a gene was found to be missing for that particularisolate.

TABLE 18 Number of indels present in isolates from group 2 analysis ofvirulence-associated genes involved in lipooligosaccharide (LOS)biosynthesis or modification. # of Non-synonymous Indels Present GeneNumber Total bp TK HS91 TK 2336 TK HS91 129PT HSM_0052 234 1 1 1 1 1 1 *HSM_0164 1569 0 0 1 0 1 0 * HSM_0204 1383 0 0 0 0 0 0 0 HSM_0397 930 0 00 0 0 0 0 HSM_0572 555 0 0 0 0 0 0 0 HSM_0840 588 0 0 0 0 0 0 0 HSM_09251431 0 0 0 0 0 0 0 HSM_0938 1167 * 0 0 0 0 0 0 HSM_0975 696 0 0 * 0 *0 * HSM_0977 834 2 0 0 0 0 0 * HSM_0978 816 0 0 1 0 1 0 * HSM_1062 13650 0 0 0 0 0 0 HSM_1063 888 0 0 0 0 0 0 0 HSM_1117 672 0 0 0 0 0 0 1HSM_1118 1020 0 0 0 0 0 0 0 HSM_1256 1017 0 0 0 0 0 0 0 HSM_1426 903 0 00 0 0 0 * HSM_1667 129 * 0 0 0 0 0 * HSM_1669 876 0 0 0 0 0 0 0 HSM_1714630 0 * * 0 * * * HSM_1794 936 0 0 0 0 0 0 * HSM_1832 1656 1 0 1 0 1 01 * Indicates when a gene was found to be missing for that particularisolate.

TABLE 19 (5) Number of indels present in isolates from group 1 analysisof virulence-associated genes involved in adhesion, colonization, orbiofilm formation. # of Non-synonymous Indels Present Gene Number Totalbp HS91 TK #21 TK #4 153-3 129PT HSM_0077 12192 0 3 2 * * HSM_0268 71190 1 1 * * HSM_0270 3480 0 * * * * HSM_0274 1569 0 1 0 * * HSM_0338 72600 * * * * HSM_0346 4653 0 3 3 * * HSM_0377 9948 0 * * * * HSM_0394 98700 1 1 * * HSM_0695 924 0 0 0 0 * HSM_0708 11025 0 * * * * HSM_0844 94170 6 6 1 * HSM_0953 1227 0 * * 1 1 HSM_1022 11250 0 4 4 1 6 HSM_1090 52680 3 3 3 * HSM_1212 1170 0 0 0 0 * HSM_1257 13971 0 * * * * HSM_1484 65400 3 4 * * HSM_1542 7158 0 6 8 * * HSM_1571 11772 0 * 4 4 * HSM_1647 21240 0 0 0 * HSM_1651 8235 0 2 1 0 * HSM_1793 8277 0 * * * * HSM_1889 21930 * * 0 * * Indicates when a gene was found to be missing for thatparticular isolate.

TABLE 20 (6) Number of indels present in isolates from group 2 analysisof virulence-associated genes involved in adhesion, colonization, orbiofilm formation. # of Non-synonymous Indels Present Gene Number GeneName Total bp TK HS91 TK 2336 TK HS91 129P HSM_007 YadA 12192 4 0 * 0 *0 * HSM_026 Filament 7119 * 0 1 0 1 0 * HSM_027 Hemaggl 3480 * 0 * 0 *0 * HSM_027 Adhesin 1569 * 0 0 0 0 0 * HSM_033 YadA 7260 3 0 5 0 5 0 *HSM_034 YadA 4653 7 0 5 0 5 0 * HSM_037 YadA 9948 5 * 8 0 8 * * HSM_039YadA 9870 0 1 0 0 0 1 * HSM_069 Alkaline 924 0 0 0 0 0 0 * HSM_070 YadA11025 * 0 * 0 * 0 * HSM_084 YadA 9417 1 0 5 0 5 0 * HSM_095 Hemaggl 12271 0 0 0 0 0 1 HSM_102 YadA 11250 * 0 7 0 7 0 7 HSM_109 Filament 5268 2 33 0 3 2 * HSM_121 Hemaggl 1170 * 0 1 0 1 0 * HSM_125 YadA 13971 * 0 *0 * 0 * HSM_148 YadA 6540 4 0 1 0 1 0 * HSM_154 YadA 7158 * 0 * 0 * 0 *HSM_157 YadA 11772 * 0 * 0 * 0 * HSM_164 FhaB 2124 0 0 0 0 0 0 * HSM_165Filament 8235 0 0 0 0 0 0 * HSM_179 YadA 8277 * * * * * 0 * HSM_188Peptidase 2193 * * 0 0 0 * * * Indicates when a gene was found to bemissing for that particular isolate.

TABLE 21 Number of indels present in isolates from group 1 analysis ofvirulence- associated genes involved in host invasion or metal uptake. #of Non-synonymous Indels Present Gene Number Total bp HS91 TK #21 TK #4153-3 129PT HSM_0047 2754 0 0 2 0 1 HSM_0749 1989 1 * * * 1 HSM_07502916 0 2 2 2 4 HSM_0931 1779 0 0 0 1 0 HSM_1089 1353 0 0 0 0 * HSM_1160537 0 0 * 0 * HSM_1168 3111 0 0 0 0 1 HSM_1176 261 0 1 1 0 2 HSM_17261548 0 0 * 0 * HSM_1730 1605 0 0 0 0 0 HSM_1962 2607 0 0 0 0 0 *Indicates when a gene was found to be missing for that particularisolate.

TABLE 22 Number of indels present in isolates from group 2 analysis ofvirulence- associated genes involved in host invasion or metal uptake. #of Non-synonymous Indels Present Gene Number Total bp TK HS91 TK 2336 TKHS91 129P HSM_0047 2754 2 0 1 0 1 0 1 HSM_0749 1989 * 0 1 1 1 0 1HSM_0750 2916 2 0 2 0 1 0 3 HSM_0931 1779 0 0 0 0 0 0 0 HSM_1089 1353 00 1 0 1 0 * HSM_1160 537 * 0 0 0 0 0 * HSM_1168 3111 0 0 0 0 0 0 1HSM_1176 261 2 0 0 0 0 0 2 HSM_1726 1548 0 * * 0 * * * HSM_1730 1605 0 00 0 0 0 0 HSM_1962 2607 0 0 0 0 0 0 0 * Indicates when a gene was foundto be missing for that particular isolate.

TABLE 23 Number of indels present in isolates from group 1 analysis ofvirulence-associated genes involved in stress response, antibioticresistance, and drug efflux. # of Non-synonymous Indels Present GeneNumber Total bp HS91 TK #21 TK #4 153-3 129PT HSM_0598 1890 0 * * 0 *HSM_1191 573 0 * * * * HSM_1448 282 0 0 0 0 * HSM_1616 981 0 * * 0 *HSM_1624 1029 1 * * * * HSM_1728 405 0 * * * * HSM_1734 624 0 0 * 0 *HSM_1736 333 0 * * * * HSM_1737 450 0 * * * * HSM_1741 399 0 * * * * *Indicates when a gene was found to be missing for that particularisolate.

TABLE 24 Number of indels present in isolates from group 2 analysis ofvirulence-associated genes involved in stress response, antibioticresistance, and drug efflux. # of Non-synonymous Indels Present GeneNumber Total bp TK HS91 TK 2336 TK HS91 129PT HSM_0598 1890 * 0 0 0 00 * HSM_1191 573 * 0 * 0 * 0 * HSM_1448 282 0 0 * 0 * 0 * HSM_1616 981 00 0 0 0 0 * HSM_1624 1029 2 2 * 1 * 2 * HSM_1728 405 * * * 0 * * *HSM_1734 624 0 * * 0 * * * HSM_1736 333 * * * 0 * * * HSM_1737 450 * * *0 * * * HSM_1741 399 * * * 0 * * * * Indicates gene was missing fromthat particular isolate.

There were many similarities for indels missing among the recentisolates and 129PT for virulence-associated genes involved in hostinvasion and metal uptake. These genes included TonB-dependent receptor(2 indels for each of TK #4 and TK #34, 1 indel for TK #42, TK #28 and129PT), TbpB (1 indel each for HS91, TK #42, 2336, TK #28, and 129PT),TbpA (2 indels for TK #21, TK #4, 153-3, TK #34, and TK #42, 3 or 4indels for 129PT (the two analyses resulted in different indel estimatesfor this isolate), and 1 indel for TK #28), TbpA2 (1 indel for 153-3),hemolysis activation/secretion protein-like protein (1 indel each for TK#42 and TK #28), TonB-dependent hemoglobin/transferrin/lactoferrinfamily receptor (1 indel for 129PT), and an outer membrane heminreceptor protein (1 indel for TK #21 and TK #4, 2 indels for TK #34 and129PT). The only gene among the stress response, antibiotic resistance,and drug efflux genes to have any indels was acyltransferase 3. Therewas 1 non-synonymous indel in HS91 and 2336, and there were 2 in TK #34,HS91 ΔaroC, and HS91 ΔnanPU.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A genetically engineered,non-naturally-occurring, attenuated Histophilus somni (H. somni),strain, wherein the attenuated strain has deletion of the genesHSM_0077, HSM_0270, HSM_0708, HSM_0975, HSM_1191, HSM_1257, HSM_1448,HSM_1542, HSM_1571, HSM_1624, HSM_1714, HSM_1726, HSM_1728, HSM_1730,HSM_1734, HSM_1736, HSM_1737, HSM_1741 and HSM_1793 to eliminate theability of the genes to express their cognate wild type gene products,relative to a reference virulent H. somni strain, and wherein theattenuated strain possesses in its genome the genes HSM_0052, HSM_0164,HSM_0268, HSM_274, HSM_0338, HSM_0346, HSM_0377, HSM_0394, HSM_0598,HSM_0695, HSM_0749, HSM_0844, HSM_0938, HSM_0977, HSM_0978, HSM_1022,HSM_1089, HSM_1090, HSM_1160, HSM_1212, HSM_1426, HSM_1484, HSM_1616,HSM_1647, HSM_1651, HSM_1667, HSM_1794, and HSM_1889 relative to thereference virulent H. somni strain.
 2. A vaccine composition comprisinga pharmaceutically acceptable vehicle, diluent or excipient and anamount of the attenuated H. somni strain of claim 1 effective to elicita protective immune response against a virulent strain of H. somni in abovine animal.
 3. The vaccine of claim 2, wherein the vaccine is in theform of a formulation for intranasal or parenteral administration. 4.The vaccine of claim 2, wherein the reference virulent strain comprisesa genomic DNA sequence, which encodes and expresses 100% of the samegenes as does a strain of H. somni having the sequence set forth in SEQID NO:
 2. 5. The vaccine of claim 2, wherein the attenuated H. somnistrain encodes and expresses at least 99% of the same genes as does anattenuated H. somni strain having in its genome the sequence of SEQ IDNO:
 4. 6. The vaccine of claim 2, wherein the attenuated H. somni strainencodes and expresses 100% of the same genes as does an attenuated H.somni strain having in its genome the sequence of SEQ ID NO:
 4. 7. Thevaccine of claim 2, wherein the attenuated H. somni strain encodes andexpresses at least 99% of the same genes as does the attenuated H. somnistrain deposited under the designation PTA-121030.
 8. The vaccine ofclaim 2, wherein the attenuated H. somni strain encodes and expresses100% of the same genes as does the attenuated H. somni strain depositedunder the designation PTA-121030.
 9. The vaccine of claim 8, wherein thecomposition is non-adjuvanted.
 10. The vaccine of claim 8, wherein thecomposition further comprises at least one additional antigen capable ofeliciting a pathogen-specific immune response in a bovine animal. 11.The vaccine of claim 10, wherein the at least one additional antigen iscapable of eliciting the immune response in the bovine animal againstbovine respiratory disease complex (BRDC), bovine respiratory syncytialvirus (BRSV), bovine viral diarrhea (BVD), bovine parainfluenza 3 (P13),infectious bovine rhinotracheitis (IBR), bovine herpesvirus-1 (BHV-1),or bluetongue disease virus (BTV).
 12. A method of eliciting aprotective immune response in a bovine animal in need thereof against asubsequent virulent H. somni infection comprising the step ofadministering to the bovine animal an effective amount of the vaccine ofclaim
 2. 13. The method of claim 12, wherein the administering is byintranasal, subcutaneous, or intramuscular route.